Biological Significance of the Suppression of Oxidative Phosphorylation in Induced Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Cheng Zhang

2017-11-01

Full Text Available We discovered that induced pluripotent stem cell (iPSC clones generated from aged tissue donors (A-iPSCs fail to suppress oxidative phosphorylation. Compared to embryonic stem cells (ESCs and iPSCs generated from young donors (Y-iPSCs, A-iPSCs show poor expression of the pluripotent stem cell-specific glucose transporter 3 (GLUT3 and impaired glucose uptake, making them unable to support the high glucose demands of glycolysis. Persistent oxidative phosphorylation in A-iPSCs generates higher levels of reactive oxygen species (ROS, which leads to excessive elevation of glutathione (a ROS-scavenging metabolite and a blunted DNA damage response. These phenotypes were recapitulated in Y-iPSCs by inhibiting pyruvate dehydrogenase kinase (PDK or supplying citrate to activate oxidative phosphorylation. In addition, oxidative phosphorylation in A-iPSC clones depletes citrate, a nuclear source of acetyl group donors for histone acetylation; this consequently alters histone acetylation status. Expression of GLUT3 in A-iPSCs recovers the metabolic defect, DNA damage response, and histone acetylation status.

Enzymic oxidation of carbon monoxide. II

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Yagi, T

1959-01-01

An enzyme which catalyzes the oxidation of carbon monoxide into carbon dioxide was obtained in a cell free state from Desulfovibrio desulfuricans. The enzyme activity was assayed manometrically by measuring the rate of gas uptake under the atmosphere of carbon monoxide in the presence of benzyl-viologen as an oxidant. The optimum pH range was 7 to 8. The activity was slightly suppressed by illumination. The enzyme was more stable than hydrogenase or formate dehydrogenase against the heat treatment, suggesting that it is a different entity from these enzymes. In the absence of an added oxidant, the enzyme preparation produced hydrogen gas under the atmosphere of carbon monoxide. The phenomenon can be explained assuming the reductive decomposition of water. 17 references, 4 figures, 2 tables.

Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria.

Science.gov (United States)

Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Koziel, Agnieszka; Majerczak, Joanna; Zoladz, Jerzy A

2015-06-01

Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling, and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25 °C) and hyperthermia (42 °C) compared to the physiological temperature of resting muscle (35 °C). For nonphosphorylating mitochondria, increasing the temperature from 25 to 42 °C led to a decrease in membrane potential, hydrogen peroxide production, and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. However, the efficiency of oxidative phosphorylation decreased, whereas the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased, with increasing assay temperature. An increase in proton leak, including uncoupling protein-mediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic defects in the oxidative phosphorylation (OXPHOS) system.

NARCIS (Netherlands)

Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.

2004-01-01

The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual

Effects of Anthropogenic Pollution on the Oxidative Phosphorylation Pathway of Hepatocytes from Natural Populations of Fundulus heteroclitus

Energy Technology Data Exchange (ETDEWEB)

Du, Xiao; Crawford, Douglas L.; Oleksiak, Marjorie F., E-mail: moleksiak@rsmas.miami.edu

2015-08-15

Highlights: • Fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Polluted fish had lower LEAK, enzyme III and enzyme IV but higher enzyme I. • Exposures to PAH and PCB only affected individuals from the reference population. - Abstract: Persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), potentially target mitochondria and cause toxicity. We compared the effects of POPs on mitochondrial respiration by measuring oxidative phosphorylation (OxPhos) metabolism in hepatocytes isolated from lab-depurated Fundulus heteroclitus from a Superfund site contaminated with PAHs (Elizabeth River VA, USA) relative to OxPhos metabolism in individuals from a relatively clean, reference population (King’s Creek VA, USA). In individuals from the polluted Elizabeth River population, OxPhos metabolism displayed lower LEAK and lower activities in complex III, complex IV, and E State, but higher activity in complex I compared to individuals from the reference King’s Creek population. To test the supposition that these differences were due to or related to the chronic PAH contamination history of the Elizabeth River population, we compared the OxPhos functions of undosed individuals from the polluted and reference populations to individuals from these populations dosed with a PAH {benzo [α] pyrene (BaP)} or a PCB {PCB126 (3,3′,4,4′,5-pentachlorobiphenyl)}, respectively. Exposure to PAH or PCB affected OxPhos in the reference King’s Creek population but had no detectable effects on the polluted Elizabeth River population. Thus, PAH exposure significantly increased LEAK, and exposure to PCB126 significantly decreased State 3, E state and complex I activity in the reference King’s Creek population. These data strongly implicate an evolved tolerance in the Elizabeth River fish where dosed

Effects of Anthropogenic Pollution on the Oxidative Phosphorylation Pathway of Hepatocytes from Natural Populations of Fundulus heteroclitus

International Nuclear Information System (INIS)

Du, Xiao; Crawford, Douglas L.; Oleksiak, Marjorie F.

2015-01-01

Highlights: • Fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Polluted fish had lower LEAK, enzyme III and enzyme IV but higher enzyme I. • Exposures to PAH and PCB only affected individuals from the reference population. - Abstract: Persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), potentially target mitochondria and cause toxicity. We compared the effects of POPs on mitochondrial respiration by measuring oxidative phosphorylation (OxPhos) metabolism in hepatocytes isolated from lab-depurated Fundulus heteroclitus from a Superfund site contaminated with PAHs (Elizabeth River VA, USA) relative to OxPhos metabolism in individuals from a relatively clean, reference population (King’s Creek VA, USA). In individuals from the polluted Elizabeth River population, OxPhos metabolism displayed lower LEAK and lower activities in complex III, complex IV, and E State, but higher activity in complex I compared to individuals from the reference King’s Creek population. To test the supposition that these differences were due to or related to the chronic PAH contamination history of the Elizabeth River population, we compared the OxPhos functions of undosed individuals from the polluted and reference populations to individuals from these populations dosed with a PAH {benzo [α] pyrene (BaP)} or a PCB {PCB126 (3,3′,4,4′,5-pentachlorobiphenyl)}, respectively. Exposure to PAH or PCB affected OxPhos in the reference King’s Creek population but had no detectable effects on the polluted Elizabeth River population. Thus, PAH exposure significantly increased LEAK, and exposure to PCB126 significantly decreased State 3, E state and complex I activity in the reference King’s Creek population. These data strongly implicate an evolved tolerance in the Elizabeth River fish where dosed

Peroxides and radiation impairment of oxidative phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Dovgii, I E; Akoev, I G

1975-09-01

An increase in the peroxidase activity of the mitochondria and a simultaneous rise in the amount of peroxide compounds, which are half lipid-like substances, are detected within the first 10 minutes after irradiation (1000 r). A mechanism of radiation impairment of oxidative phosphorylation is connected with the penetration of its inhibitors to the mitochondria due to the disturbed permeability of membranes affected by peroxides.

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

Science.gov (United States)

Villalobo, A; Lehninger, A L

1980-03-25

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

Expression of oxidative phosphorylation components in mitochondria of long-living Ames dwarf mice.

Science.gov (United States)

Brown-Borg, Holly M; Johnson, W Thomas; Rakoczy, Sharlene G

2012-02-01

Reduced signaling of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) pathway is associated with extended life span in several species. Ames dwarf mice are GH-deficient and live >50% longer than wild-type littermates. Previously, we have shown that tissues from Ames mice exhibit elevated levels of antioxidative enzymes, less H(2)O(2) production, and lower oxidative damage suggesting that mitochondrial function may differ between genotypes. To explore the relationship between hormone deficiency and mitochondria in mice with extended longevity, we evaluated activity, protein, and gene expression of oxidative phosphorylation components in dwarf and wild-type mice at varying ages. Liver complex I + III activity was higher in dwarf mice compared to wild-type mice. The activity of I + III decreased between 3 and 20 months of age in both genotypes with greater declines in wild-type mice in liver and skeletal muscle. Complex IV activities in the kidney were elevated in 3- and 20-month-old dwarf mice relative to wild-type mice. In Ames mice, protein levels of the 39 kDa complex I subunit were elevated at 20 months of age when compared to wild-type mouse mitochondria for every tissue examined. Kidney and liver mitochondria from 20-month-old dwarf mice had elevated levels of both mitochondrially-encoded and nuclear-encoded complex IV proteins compared to wild-type mice (p dwarf mice. Overall, we found that several components of the oxidative phosphorylation (OXPHOS) system were elevated in Ames mice. Mitochondrial to nuclear DNA ratios were not different between genotypes despite the marked increase in PGC-1α levels in dwarf mice. The increased OXPHOS activities, along with lower ROS production in dwarf mice, predict enhanced mitochondrial function and efficiency, two factors likely contributing to long-life in Ames mice.

Heritable oxidative phosphorylation differences in a pollutant resistant Fundulus heteroclitus population

International Nuclear Information System (INIS)

Du, Xiao; Crawford, Douglas L.; Nacci, Diane E.; Oleksiak, Marjorie F.

2016-01-01

Highlights: • Laboratory reared fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Laboratory reared F3 fish from polluted population displayed higher routine metabolism and complex II activity but lower complex I enzyme activity. • Enhanced OxPhos metabolism and toxicity resistance were retained in laboratory reared F3 fish from the polluted population. - Abstract: Populations can adapt to stress including recent anthropogenic pollution. Our published data suggests heritable differences in hepatocyte oxidative phosphorylation (OxPhos) metabolism in field-caught killifish (Fundulus heteroclitus) from the highly polluted Elizabeth River, VA, USA, relative to fish from a nearby, relatively unpolluted reference site in King’s Creek VA. Consistent with other studies showing that Elizabeth River killifish are resistant to some of the toxic effects of certain contaminants, OxPhos measurements in hepatocytes from field-caught King’s Creek but not field-caught Elizabeth River killifish were altered by acute benzo [a] pyrene exposures. To more definitively test whether the enhanced OxPhos metabolism and toxicity resistance are heritable, we measured OxPhos metabolism in a laboratory-reared F3 generation from the Elizabeth River population versus a laboratory-reared F1 generation from the King’s Creek population and compared these results to previous data from the field-caught fish. The F3 Elizabeth River fish compared to F1 King’s Creek fish had significantly higher State 3 respiration (routine metabolism) and complex II activity, and significantly lower complex I activity. The consistently higher routine metabolism in the F3 and field-caught Elizabeth River fish versus F1 and field-caught King’s Creek fish implies a heritable change in OxPhos function. The observation that LEAK, E-State, Complex I and Complex II were different in laboratory bred

Heritable oxidative phosphorylation differences in a pollutant resistant Fundulus heteroclitus population

Energy Technology Data Exchange (ETDEWEB)

Du, Xiao, E-mail: xdu@rsmas.miami.edu [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States); Crawford, Douglas L. [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States); Nacci, Diane E. [Population Ecology Branch, Atlantic Ecology Division, Office of Research and Development, U.S. Environmental Protection Agency, 27 Tarzwell Dr., Narragansett, RI 02882 (United States); Oleksiak, Marjorie F., E-mail: moleksiak@rsmas.miami.edu [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States)

2016-08-15

Highlights: • Laboratory reared fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Laboratory reared F3 fish from polluted population displayed higher routine metabolism and complex II activity but lower complex I enzyme activity. • Enhanced OxPhos metabolism and toxicity resistance were retained in laboratory reared F3 fish from the polluted population. - Abstract: Populations can adapt to stress including recent anthropogenic pollution. Our published data suggests heritable differences in hepatocyte oxidative phosphorylation (OxPhos) metabolism in field-caught killifish (Fundulus heteroclitus) from the highly polluted Elizabeth River, VA, USA, relative to fish from a nearby, relatively unpolluted reference site in King’s Creek VA. Consistent with other studies showing that Elizabeth River killifish are resistant to some of the toxic effects of certain contaminants, OxPhos measurements in hepatocytes from field-caught King’s Creek but not field-caught Elizabeth River killifish were altered by acute benzo [a] pyrene exposures. To more definitively test whether the enhanced OxPhos metabolism and toxicity resistance are heritable, we measured OxPhos metabolism in a laboratory-reared F3 generation from the Elizabeth River population versus a laboratory-reared F1 generation from the King’s Creek population and compared these results to previous data from the field-caught fish. The F3 Elizabeth River fish compared to F1 King’s Creek fish had significantly higher State 3 respiration (routine metabolism) and complex II activity, and significantly lower complex I activity. The consistently higher routine metabolism in the F3 and field-caught Elizabeth River fish versus F1 and field-caught King’s Creek fish implies a heritable change in OxPhos function. The observation that LEAK, E-State, Complex I and Complex II were different in laboratory bred

Subunit topology in the V type ATPase and related enzymes

NARCIS (Netherlands)

Chaban, Yuriy

2005-01-01

During the last decades impressive progress has been made in understanding of the catalytic mechanism of F-type ATP synthase, which is the key enzyme in the energy metabolism of eukaryotes and most bacteria. This enzyme catalyzes the final step in the process of oxidative phosphorylation in bacteria

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Mahalingan, Krishna K.; Baskaran†, Sulochanadevi; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med)

2017-01-10

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

Electrical stimulation affects metabolic enzyme phosphorylation, protease activation and meat tenderization in beef

DEFF Research Database (Denmark)

Li, C.B.; Li, J.; Zhou, G.H.

2012-01-01

The objective of this study was to investigate the response of sarcoplasmic proteins in bovine longissimus muscle to low-voltage electrical stimulation (ES, 80 V, 35 s) after dressing and its contribution to meat tenderization at early postmortem time. Proteome analysis showed that ES resulted...... muscles up to 24 h. Immunohistochemistry and transmission electron microscopy further indicated that lysosomal enzymes were released at early postmortem time. ES also induced ultrastructural disruption of sarcomeres. In addition, ES accelerated (P ..., as well as pH decline and more preferred pH/temperature decline mode. Finally, ES accelerated meat tenderization with lower (P time. A possible relationship was suggested between change in phosphorylation level of energy metabolic enzymes and postmortem...

Aluminum coordination chemistry and the inhibition of phosphoryl-transferring enzymes

International Nuclear Information System (INIS)

Furumo, N.C.; Viola, R.E.

1986-01-01

Aluminium ion is a potent inhibitor of the enzymes hexokinase (K/sub i/ = 0.16 μM) and glycerokinase (K/sub i/ = 4.0 μM). It has been shown that aluminum forms a complex with ATP that is 80 times more stable than the magnesium complex with ATP which is the normal substrate for phosphoryl-transferring enzymes. Kinetic studies performed on several kinases at pH 7.0 have shown that Al-ATP is a competitive inhibitor vs. Mg-ATP with moderate K/sub i/ values (0.1-0.5 mM) for creatine kinase(CK) and myokinase(MK), and weakly competitive (K/sub i/ > 0.5 mM) with acetate, galactose, arginine and gluconate kinases. Equilibrium dialysis binding studies indicate no significant binding of aluminum ion by the enzymes, while the interaction of aluminum ion with ADP and ATP has been characterized by 13 C, 27 Al, and 31 P NMR spectroscopy. It appears that the inhibition by aluminum is as the Al-nucleotide complex rather than direct binding of free aluminum ion by the enzyme. Kinetic studies indicate that Al 3+ inhibition of CK and MK is pH dependent with decreased values of K/sub i/ at lower pH. At pH 6.1 K/sub i/ = 25 μM for MK (160 μM at pH 7.0) and 53 μM for CK (240 μM at pH 7.0). This may be due to an increased effective concentration of aluminum ion at lower pH

BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells

Science.gov (United States)

Lagadinou, Eleni D.; Sach, Alexander; Callahan, Kevin; Rossi, Randall M.; Neering, Sarah J.; Minhajuddin, Mohammad; Ashton, John M.; Pei, Shanshan; Grose, Valerie; O’Dwyer, Kristen M.; Liesveld, Jane L.; Brookes, Paul S.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

Summary Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally-defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly over-express BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation. PMID:23333149

Coordination of manganous ion at the active site of pyruvate, phosphate dikinase: the complex of oxalate with the phosphorylated enzyme

International Nuclear Information System (INIS)

Kofron, J.L.; Ash, D.E.; Reed, G.H.

1988-01-01

Electron paramagnetic resonance spectroscopy has been used to investigate the structure of the complex of manganous ion with the phosphorylated form of pyruvate, phosphate dikinase (E/sub p/) and the inhibitor oxalate. Oxalate, an analogue of the enolate of pyruvate, is competitive with respect to pyruvate in binding to the phosphorylated form of the enzyme. Superhyperfine coupling between the unpaired electrons of Mn(I) and ligands specifically labeled with 17 O has been used to identify oxygen ligands to Mn(II) in the complex with oxalate and the phosphorylated form of the enzyme. Oxalate binds at the active site as a bidentate chelate with Mn(II). An oxygen from the 3'-N-phosphohistidyl residue of the protein is in the coordination sphere of Mn(II), and at least two water molecules are also bound to Mn(II) in the complex. Oxalate also binds directly to Mn(II) in a complex with nonphosphorylated enzyme. The structure for the E/sub p/-Mn(II)-oxalate complex implies that simultaneous coordination of a phospho group and of the attacking nucleophile to the divalent cation is likely an important factor in catalysis of this phospho-transfer reaction

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

Science.gov (United States)

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

OpenAIRE

Korzeniewski, Bernard; Zoladz, Jerzy A

2003-01-01

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial protein...

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Science.gov (United States)

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, S P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2011-09-01

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

Direct evidence for the inactivation of branched-chain oxo-acid dehydrogenase by enzyme phosphorylation

International Nuclear Information System (INIS)

Odessey, R.

1980-01-01

The branched-chain 2-oxo-acid dehydrogenase (BCOAD) from mitochondria of several different rat tissues is inactivated by ATP and can be reactivated by incubation in Mg 2+ -containing buffers. Work carried out on the system from skeletal muscle mitochondria has shown that inactivation requires the cleavage of the γ-phosphate group of ATP and that modification is covalent. The non-metabolized ATP analog, p[NH]ppA, can block the inhibitory effect of ATP when added prior to ATP addition, but cannot reverse the inhibition of the inactivated dehydrogenase. These and other data raise the possibility that BCOAD may be regulated by enzyme phosphorylation. This hypothesis is supported by the finding that various procedures which separate the enzyme from its mitochondrial environment (e.g. detergent treatment, ammonium sulfate precipitation and freeze-thawing) do not alter the degree of inhibition induced by ATP in the mitochondrial preincubation. These experiments suggested the feasibility of labelling the enzyme with 32 P and purifying it. (Auth.)

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

Science.gov (United States)

Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

OpenAIRE

Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

1990-01-01

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

Xenobiotics that affect oxidative phosphorylation alter differentiation of human adipose-derived stem cells at concentrations that are found in human blood

Directory of Open Access Journals (Sweden)

Laura Llobet

2015-11-01

Full Text Available Adipogenesis is accompanied by differentiation of adipose tissue-derived stem cells to adipocytes. As part of this differentiation, biogenesis of the oxidative phosphorylation system occurs. Many chemical compounds used in medicine, agriculture or other human activities affect oxidative phosphorylation function. Therefore, these xenobiotics could alter adipogenesis. We have analyzed the effects on adipocyte differentiation of some xenobiotics that act on the oxidative phosphorylation system. The tested concentrations have been previously reported in human blood. Our results show that pharmaceutical drugs that decrease mitochondrial DNA replication, such as nucleoside reverse transcriptase inhibitors, or inhibitors of mitochondrial protein synthesis, such as ribosomal antibiotics, diminish adipocyte differentiation and leptin secretion. By contrast, the environmental chemical pollutant tributyltin chloride, which inhibits the ATP synthase of the oxidative phosphorylation system, can promote adipocyte differentiation and leptin secretion, leading to obesity and metabolic syndrome as postulated by the obesogen hypothesis.

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

Science.gov (United States)

Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

2016-01-01

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

Science.gov (United States)

Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

Effective reaction rates in diffusion-limited phosphorylation-dephosphorylation cycles

Science.gov (United States)

Szymańska, Paulina; Kochańczyk, Marek; Miekisz, Jacek; Lipniacki, Tomasz

2015-02-01

We investigate the kinetics of the ubiquitous phosphorylation-dephosphorylation cycle on biological membranes by means of kinetic Monte Carlo simulations on the triangular lattice. We establish the dependence of effective macroscopic reaction rate coefficients as well as the steady-state phosphorylated substrate fraction on the diffusion coefficient and concentrations of opposing enzymes: kinases and phosphatases. In the limits of zero and infinite diffusion, the numerical results agree with analytical predictions; these two limits give the lower and the upper bound for the macroscopic rate coefficients, respectively. In the zero-diffusion limit, which is important in the analysis of dense systems, phosphorylation and dephosphorylation reactions can convert only these substrates which remain in contact with opposing enzymes. In the most studied regime of nonzero but small diffusion, a contribution linearly proportional to the diffusion coefficient appears in the reaction rate. In this regime, the presence of opposing enzymes creates inhomogeneities in the (de)phosphorylated substrate distributions: The spatial correlation function shows that enzymes are surrounded by clouds of converted substrates. This effect becomes important at low enzyme concentrations, substantially lowering effective reaction rates. Effective reaction rates decrease with decreasing diffusion and this dependence is more pronounced for the less-abundant enzyme. Consequently, the steady-state fraction of phosphorylated substrates can increase or decrease with diffusion, depending on relative concentrations of both enzymes. Additionally, steady states are controlled by molecular crowders which, mostly by lowering the effective diffusion of reactants, favor the more abundant enzyme.

Systematic inference of functional phosphorylation events in yeast metabolism.

Science.gov (United States)

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures

International Nuclear Information System (INIS)

Ray, Bruce D.; Jarori, Gotam K.; Nageswara Rao, B.D.

2002-01-01

31 P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, E·CoATP·arginine ↔ E·CoADP·P-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289-293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)- 31 P (γ-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)- 31 P distance of ∼3.0 A, representing direct coordination of Co(II) to γ-P(ATP), changes to ∼4.0 A when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)- 31 P vector implies an excursion of at least 1.0 A for the itinerant phosphoryl group on the surface of the enzyme

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches.

Science.gov (United States)

Wakai, Satoshi; Masanari, Misa; Ikeda, Takumi; Yamaguchi, Naho; Ueshima, Saori; Watanabe, Kaori; Nishihara, Hirofumi; Sambongi, Yoshihiro

2013-04-01

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

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Masahiro Myojo

Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion.

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Lawton, Thomas J; Rosenzweig, Amy C

2016-08-03

Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16-13 s(-1), these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock.

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1 preserves oxidative phosphorylation in a yeast model of Barth syndrome

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Maxence de Taffin de Tilques

2017-04-01

Full Text Available Cardiolipin (CL is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS. To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS, presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1Δ yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS.

CELLULOSE DEGRADATION BY OXIDATIVE ENZYMES

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Maria Dimarogona

2012-09-01

Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

Scaling of oxidative and glycolytic enzymes in mammals.

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Emmett, B; Hochachka, P W

1981-09-01

The catalytic activities of several oxidative and glycolytic enzymes were determined in the gastrocnemius muscle of 10 mammalian species differing in body weight by nearly 6 orders of magnitude. When expressed in terms of units gm-1, the activities of enzymes functioning in oxidative metabolism (citrate synthase, beta-hydroxybutyrylCoA dehydrogenase, and malate dehydrogenase) decrease as body weight increases. Log-log plots (activity gm-1 vs body mass) yield straight lines with negative slopes that are less than the allometric exponent (-0.25) typically observed for basal metabolic rates. Since the amount of power a muscle can generate depends upon the catalytic potential of its enzyme machinery (the higher the catalytic potential the higher the maximum rate of energy generation), these data predict that the scope for aerobic activity in large mammals should be greater than in small mammals if nothing else becomes limiting, a result in fact recently obtained by Taylor et al. (Respir. Physiol., 1981). In contrast to the scaling of oxidative enzymes, the activities of enzymes functioning in anaerobic glycogenolysis (glycogen phosphorylase, pyruvate kinase, and lactate dehydrogenase) increase as body size increases. Log-log plots (activity gm-1 vs body mass) display a positive slope indicating that the larger the animal the higher the glycolytic potential of its skeletal muscles. This unexpected result may indicate higher relative power costs for burst type locomotion in larger mammals, which is in fact observed in within-species studies of man. However, the scaling of anaerobic muscle power has not been closely assessed in between-species comparisons of mammals varying greatly in body size.

Parkinson's disease associated with impaired oxidative phosphorylation

International Nuclear Information System (INIS)

Finsterer, J.; Jarius, C.; Baumgartner, M.

2001-01-01

Parkinson's disease may be due to primary or secondary oxidative phosphorylation (OXPHOS) defects. In a 76-year-old man with Parkinson's disease since 1992, slightly but recurrently elevated creatine phosphokinase, recurrently elevated blood glucose, thickening of the left ventricular myocardium, bifascicular block and hypacusis were found. Cerebral MRI showed atrophy, periventricular demyelination, multiple, disseminated, supra- and infratentorial lacunas, and haemosiderin deposits in both posterior horns. Muscle biopsy showed typical features of an OXPHOS defect. Whether the association of Parkinson's disease and impaired OXPHOS was causative or coincidental remains unknown. Possibly, the mitochondrial defect acted as an additional risk factor for Parkinson's disease or the OXPHOS defect worsened the preexisting neurological impairments by a cumulative or synergistic mechanism. In conclusion, this case shows that Parkinson's disease may be associated with a mitochondrially or nuclearly encoded OXPHOS defect, manifesting as hypacusis, myopathy, axonal polyneuropathy, cardiomyopathy and recurrent subclinical ischaemic strokes and haemorrhages. (orig.)

Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato

NARCIS (Netherlands)

Bultema, Jelle B.; Braun, Hans-Peter; Boekema, Egbert J.; Kouřil, Roman

The individual protein complexes of the oxidative phosphorylation system (OXPHOS complexes 1 to V) specifically interact and form defined supramolecular structures, the so-called "respiratory supercomplexes". Some supercomplexes appear to associate into larger structures, or megacomplexes, such as a

Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: {sup 31}P relaxation measurements on enzyme-bound equilibrium mixtures

Energy Technology Data Exchange (ETDEWEB)

Ray, Bruce D. [Indiana University, Purdue University at Indianapolis (IUPUI), Department of Physics (United States); Jarori, Gotam K. [Tata Institute of Fundamental Research (India); Nageswara Rao, B.D. [Indiana University, Purdue University at Indianapolis (IUPUI), Department of Physics (United States)], E-mail: brao@iupui.edu

2002-05-15

{sup 31}P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, E{center_dot}CoATP{center_dot}arginine {r_reversible} E{center_dot}CoADP{center_dot}P-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289-293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-{sup 31}P ({gamma}-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-{sup 31}P distance of {approx}3.0 A, representing direct coordination of Co(II) to {gamma}-P(ATP), changes to {approx}4.0 A when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-{sup 31}P vector implies an excursion of at least 1.0 A for the itinerant phosphoryl group on the surface of the enzyme.

Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria.

Science.gov (United States)

Filipović, Dragana; Costina, Victor; Perić, Ivana; Stanisavljević, Andrijana; Findeisen, Peter

2017-03-15

Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics. Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

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Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Factors influencing radiation-induced impairment of rat liver mitochondrial oxidative phosphorylation

International Nuclear Information System (INIS)

Alexander, K.C.; Aiyar, A.S.; Sreenivasan, A.

1975-01-01

The influence of some experimental conditions on the radiation-induced impairment of oxidative phosphorylation in rat liver mitochondria has been studied. Shielding of the liver during whole body irradiation of the animal does not significantly alter the decreased efficiency of phosphorylation. There exists a great disparity in the in vivo and in vitro radiation doses required for the manifestation of damage to liver mitochondria. While these observations point to the abscopal nature of the radiation effects, direct involvement of the adrenals has been ruled out by studies with adrenalectomised rats. Prior administration of the well known radio-protective agents, serotonin or 2-aminoethyl isothiouronium bromide hydrobromide, is effective in preventing the derangement of mitochondrial function following radioexposure. The hypocholesterolemic drug ethyl-α-p-chlorophenoxy isobutyrate, which is known to influence hepatic mitochondrial turnover, does not afford any significant protection against either mitochondrial damage or the mortality of the animals due to whole body irradiation. (author)

Hepatic fatty acid oxidation : activity, localization and function of some enzymes involved

NARCIS (Netherlands)

A. van Tol (Arie)

1971-01-01

textabstractFatty acid oxidation is an important pathway for energy production in mammals and birds. In animal tissues the enzymes of fatty acid oxidation are located in the mitochondrion. Recent reports suggest that this is not the case in Castor bean endosperm. In this tissue the enzymes of

Early bichemical markers of effects: Enzyme induction, oncogene activation and markers of oxidative damage

DEFF Research Database (Denmark)

Poulsen, Henrik E.; Loft, Steffen

1995-01-01

Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein......Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein...

Expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and mitochondrial oxidative phosphorylation in septic patients.

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Laura A Nucci

Full Text Available Sepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients.Blood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0, and after 7 days (D7, N = 10 of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant.Twenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients.Our data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by changes in JAK-STAT modulation, had prominent changes in

Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress

Energy Technology Data Exchange (ETDEWEB)

Muhsain, Siti Nur Fadzilah, E-mail: sitinurfadzilah077@ppinang.uitm.edu.my [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Faculty of Pharmacy, University Teknologi Mara (Malaysia); Lang, Matti A., E-mail: m.lang@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

2015-01-01

The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200 mg pyrazole/kg/day for 3 days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. - Highlights: • Pyrazole induces oxidative stress in the mouse liver. • Pyrazole-induced oxidative stress induces mitochondrial targeting of key bilirubin regulatory enzymes, HMOX1

Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress

International Nuclear Information System (INIS)

Muhsain, Siti Nur Fadzilah; Lang, Matti A.; Abu-Bakar, A'edah

2015-01-01

The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200 mg pyrazole/kg/day for 3 days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. - Highlights: • Pyrazole induces oxidative stress in the mouse liver. • Pyrazole-induced oxidative stress induces mitochondrial targeting of key bilirubin regulatory enzymes, HMOX1

IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION

Science.gov (United States)

IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION.T. L. Knuckles1 R. Jaskot2, J. Richards2, and K.Dreher2.1Department of Molecular and Biomedical Sciences, College of Veterinary Medicin...

Photobiomodulation Therapy Decreases Oxidative Stress in the Lung Tissue after Formaldehyde Exposure: Role of Oxidant/Antioxidant Enzymes

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Rodrigo Silva Macedo

2016-01-01

Full Text Available Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days and treated or not with PBMT (1 and 5 h after each FA exposure. Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment.

Regulation of oxidative enzyme activity and eukaryotic elongation factor 2 in human skeletal muscle: influence of gender and exercise

DEFF Research Database (Denmark)

Roepstorff, Carsten; Schjerling, P.; Vistisen, Bodil

2005-01-01

AIM: To investigate gender-related differences in the responses of oxidative enzymes and eukaryotic elongation factor-2 (eEF2) to exercise. METHODS: The influence of exercise (90 min, 60%VO(2peak)) on citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HAD) activity and mRNA content...... expression and phosphorylation were unaffected by training status (NS). CONCLUSION: Basal transcriptional, translational, and/or post-translational control of CS and HAD seems to be gender-dependent. Also, gender differences in translation and/or post-translational protein modification of CS occur during...... not differ between females and males (NS). In females only, CS activity was enhanced (P differ between UT and ET but, nevertheless, CS activity was 56% higher in ET than in UT volunteers (P

Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

Science.gov (United States)

Jo-Watanabe, Airi; Ohse, Takamoto; Nishimatsu, Hiroaki; Takahashi, Masao; Ikeda, Yoichiro; Wada, Takehiko; Shirakawa, Jun-ichi; Nagai, Ryoji; Miyata, Toshio; Nagano, Tetsuo; Hirata, Yasunobu; Inagi, Reiko; Nangaku, Masaomi

2014-06-01

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons

Mitochondrial oxidative function and type 2 diabetes

DEFF Research Database (Denmark)

Rabøl, Rasmus; Boushel, Robert; Dela, Flemming

2006-01-01

The cause of insulin resistance and type 2 diabetes is unknown. The major part of insulin-mediated glucose disposal takes place in the skeletal muscle, and increased amounts of intramyocellular lipid has been associated with insulin resistance and linked to decreased activity of mitochondrial...... oxidative phosphorylation. This review will cover the present knowledge and literature on the topics of the activity of oxidative enzymes and the electron transport chain (ETC) in skeletal muscle of patients with type 2 diabetes. Different methods of studying mitochondrial function are described, including...... biochemical measurements of oxidative enzyme and electron transport activity, isolation of mitochondria for measurements of respiration, and ATP production and indirect measurements of ATP production using nuclear magnetic resonance (NMR) - spectroscopy. Biochemical markers of mitochondrial content are also...

Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress

Directory of Open Access Journals (Sweden)

Claudia Rodrigues

2016-12-01

Full Text Available Aquaporin-5 (AQP5 is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5’s ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics.

DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

Science.gov (United States)

Pettigrew, Christopher A; Clerkin, John S; Cotter, Thomas G

2012-12-01

Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

A novel redox-based switch: LMW-PTP oxidation enhances Grb2 binding and leads to ERK activation

International Nuclear Information System (INIS)

Giannoni, Elisa; Raugei, Giovanni; Chiarugi, Paola; Ramponi, Giampietro

2006-01-01

Low molecular weight-PTP has been reported as a redox-sensitive protein during both platelet-derived growth factor and integrin signalling. In response to oxidation the phosphatase undergoes a reversible inactivation, which in turn leads to the increase in tyrosine phosphorylation of its substrates and the properly executed anchorage-dependent proliferation program. Here, we report that an exogenous oxidative stress enhances LMW-PTP tyrosine phosphorylation, through oxidation/inactivation of the enzyme, thus preventing its auto-dephosphorylation activity. In particular, we observed a selective hyper-phosphorylation of Tyr132, that acts as a docking site for the adaptor protein Grb2. The redox-dependent enhancement of Grb2 recruitment to LMW-PTP ultimately leads to an improvement of ERK activation, likely triggering a prosurvival signal against the oxidant environment

Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis.

Science.gov (United States)

Chen, Yanmei; Hoehenwarter, Wolfgang

2015-12-01

Salinity and oxidative stress are major factors affecting and limiting the productivity of agricultural crops. The molecular and biochemical processes governing the plant response to abiotic stress have often been researched in a reductionist manner. Here, we report a systemic approach combining metabolic labeling and phosphoproteomics to capture early signaling events with quantitative metabolome analysis and enzyme activity assays to determine the effects of salt and oxidative stress on plant physiology. K(+) and Na(+) transporters showed coordinated changes in their phosphorylation pattern, indicating the importance of dynamic ion homeostasis for adaptation to salt stress. Unique phosphorylation sites were found for Arabidopsis (Arabidopsis thaliana) SNF1 kinase homolog10 and 11, indicating their central roles in the stress-regulated responses. Seven Sucrose Non-fermenting1-Related Protein Kinase2 kinases showed varying levels of phosphorylation at multiple serine/threonine residues in their kinase domain upon stress, showing temporally distinct modulation of the various isoforms. Salinity and oxidative stress also lead to changes in protein phosphorylation of proteins central to photosynthesis, in particular the kinase State Transition Protein7 required for state transition and light-harvesting II complex proteins. Furthermore, stress-induced changes of the phosphorylation of enzymes of central metabolism were observed. The phosphorylation patterns of these proteins were concurrent with changes in enzyme activity. This was reflected by altered levels of metabolites, such as the sugars sucrose and fructose, glycolysis intermediates, and amino acids. Together, our study provides evidence for a link between early signaling in the salt and oxidative stress response that regulates the state transition of photosynthesis and the rearrangement of primary metabolism. © 2015 American Society of Plant Biologists. All Rights Reserved.

Reliability of maximal mitochondrial oxidative phosphorylation in permeabilized fibers from the vastus lateralis employing high-resolution respirometry

DEFF Research Database (Denmark)

Cardinale, Daniele A; Gejl, Kasper D; Ørtenblad, Niels

2018-01-01

The purpose was to assess the impact of various factors on methodological errors associated with measurement of maximal oxidative phosphorylation (OXPHOS) in human skeletal muscle determined by high-resolution respirometry in saponin-permeabilized fibers. Biopsies were collected from 25 men...

Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

International Nuclear Information System (INIS)

Smiley, R.M.; Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J.

1990-01-01

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32 PO 4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M r 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

Hydrocortisone and insulin effect on oxidative phosphorylation in mitochondria of liver and spleen th rats exposed to fast neutrons

International Nuclear Information System (INIS)

Sutkovoj, D.A.; Alferov, A.N.; Letov, V.N.

1979-01-01

Total-body exposure of rats to fast neutrons (100 rad) elicits an increase in the content of 11-oxycorticosteroids in blood plasma and an appreciable decrease in the respiratory and phosphorylation activity of liver and spleen mitochondria. Administration of hydrocortisone (50 mg/kg) and particularly insulin (2 units/kg) intensifies energy generation in the liver. In the spleen, a beneficial effect was only produced by insulin. Glucocorticoid considerably aggravates the postradiation impairment of oxidative phosphorylation

The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions.

Science.gov (United States)

Daniel, Bastian; Konrad, Barbara; Toplak, Marina; Lahham, Majd; Messenlehner, Julia; Winkler, Andreas; Macheroux, Peter

2017-10-15

Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B 2 -derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mitochondrial oxidative phosphorylation efficiency is upregulated during fasting in two major oxidative tissues of ducklings.

Science.gov (United States)

Monternier, Pierre-Axel; Teulier, Loïc; Drai, Jocelyne; Bourguignon, Aurore; Collin-Chavagnac, Delphine; Hervant, Frédéric; Rouanet, Jean-Louis; Roussel, Damien

2017-10-01

Fasted endothermic vertebrates must develop physiological responses to maximize energy conservation and survival. The aim of this study was to determine the effect of 1-wk. fasting in 5-wk. old ducklings (Cairina moschata) from whole-body resting metabolic rate and body temperature to metabolic phenotype of tissues and mitochondrial coupling efficiency. At the level of whole organism, the mass-specific metabolic rate of ducklings was decreased by 40% after 1-wk. of fasting, which was associated with nocturnal Tb declines and shallow diurnal hypothermia during fasting. At the cellular level, fasting induced a large reduction in liver, gastrocnemius (oxidative) and pectoralis (glycolytic) muscle masses together with a fuel selection towards lipid oxidation and ketone body production in liver and a lower glycolytic phenotype in skeletal muscles. At the level of mitochondria, fasting induced a reduction of oxidative phosphorylation activities and an up-regulation of coupling efficiency (+30% on average) in liver and skeletal muscles. The present integrative study shows that energy conservation in fasted ducklings is mainly achieved by an overall reduction in mitochondrial activity and an increase in mitochondrial coupling efficiency, which would, in association with shallow hypothermia, increase the conservation of endogenous fuel stores during fasting. Copyright © 2017 Elsevier Inc. All rights reserved.

Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

Science.gov (United States)

Guntur, Anyonya R; Gerencser, Akos A; Le, Phuong T; DeMambro, Victoria E; Bornstein, Sheila A; Mookerjee, Shona A; Maridas, David E; Clemmons, David E; Brand, Martin D; Rosen, Clifford J

2018-06-01

Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK-3β

Energy Technology Data Exchange (ETDEWEB)

Song, Jae-Sook; Kim, Eun-Kyung; Choi, Yong-Won [Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588 (Korea, Republic of); Oh, Won Keun [College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University (Korea, Republic of); Kim, Young-Mi, E-mail: ymikim12@hanyang.ac.kr [Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588 (Korea, Republic of)

2016-09-15

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level. Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3β through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3β inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3β signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant

PhosphoBase: a database of phosphorylation sites

DEFF Research Database (Denmark)

Blom, Nikolaj; Kreegipuu, Andres; Brunak, Søren

1998-01-01

PhosphoBase is a database of experimentally verified phosphorylation sites. Version 1.0 contains 156 entries and 398 experimentally determined phosphorylation sites. Entries are compiled and revised from the literature and from major protein sequence databases such as SwissProt and PIR. The entries...... provide information about the phosphoprotein and the exact position of its phosphorylation sites. Furthermore, part of the entries contain information about kinetic data obtained from enzyme assays on specific peptides. To illustrate the use of data extracted from PhosphoBase we present a sequence logo...... displaying the overall conservation of positions around serines phosphorylated by protein kinase A (PKA). PhosphoBase is available on the WWW at http://www.cbs.dtu.dk/databases/PhosphoBase/....

Hepatic mitochondrial oxidative phosphorylation is normal in obese patients with and without type 2 diabetes

DEFF Research Database (Denmark)

Lund, Michael Taulo; Kristensen, Marianne Dalsgaard; Hansen, Merethe

2016-01-01

INTRODUCTION: Obese patients with (T2DM) and without (OB) type 2 diabetes are characterized by high hepatic lipid content and hepatic insulin resistance. This may be linked to impaired hepatic mitochondrial oxidative phosphorylation (OXPHOS) capacity. The aim of the present study was to investiga...... role in the development of obesity-induced type 2 diabetes. This article is protected by copyright. All rights reserved....

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

Directory of Open Access Journals (Sweden)

Javier Modrego

Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea.

Science.gov (United States)

Tolar, Bradley B; Herrmann, Jonathan; Bargar, John R; van den Bedem, Henry; Wakatsuki, Soichi; Francis, Christopher A

2017-10-01

Knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowledge of key N-cycling enzymes from ecologically relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, through (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) and nitrite reductase (NirK). © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Postmortem Changes in Pork Muscle Protein Phosphorylation in Relation to the RN Genotype

DEFF Research Database (Denmark)

Lametsch, René; Larsen, Martin Røssel; Essén-Gustavsson, Birgitta

2011-01-01

Postmortem changes in pork muscle protein phosphorylation in relation to the RN(-) genotype were investigated using one-dimensional gel electrophoresis and a phosphor specific staining. The phosphorylation levels of several protein bands were found to be affected by the RN(-) genotype and to change...... of phosphorylation of these key enzymes during the postmortem metabolism. The results illustrate that the protein phosphorylation level of the muscle proteins could be interpreted as a global metabolic fingerprint containing information about the activity status of the enzymes in the postmortem metabolism....... during postmortem development. Glycogen phosphorylase, phosphofructokinase, and pyruvate kinase were found in protein bands affected by the RN(-) genotype, and the phosphorylation profile indicates that part of the increased rate and extended pH decline of the RN(-) genotype could be a consequence...

Threonine phosphorylation of rat liver glycogen synthase

International Nuclear Information System (INIS)

Arino, J.; Arro, M.; Guinovart, J.J.

1985-01-01

32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

Energy Technology Data Exchange (ETDEWEB)

Rajagopal, P.; Klevit, R.E. [Univ. of Washington, Seattle, WA (United States)

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residue at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.

Flux control through protein phosphorylation in yeast

DEFF Research Database (Denmark)

Chen, Yu; Nielsen, Jens

2016-01-01

Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

Study of oxidative and phosphorylative activity in mitochondria from cereal seedlings

Energy Technology Data Exchange (ETDEWEB)

Plhak, F

1973-01-01

In the present paper the oxidative and phosphorylative activity of mitochondria isolated from rye, wheat, barley and corn seedlings are compared. Mitochondria from the shoots as well as from the roots of rye, wheat and corn oxidized succinate and in the presence of ATP or ADP exhibited the respiratory control which reached the values mostly of about 2. In the presence of ATP or ADP the decrease of inorganic phosphorus was contemporarily remarkable. The P/O ratio reached the values mostly of about 0.8 up to 1.0. The presence of ATP effected in some cases more favorable the respiratory control as well as the P/O ratio in comparison with ADP. With regard to the fact that a trapping hexokinase system was not added, the presence of endogenous hexokinase in mitochondria of experimental plants is presumed. The barley mitochondria exhibited the respiratory control as well, but instead of the decrease in inorganic phosphorus content in reaction mixture, an increase took place. It was caused by the presence of active ATP-ase which was not effectively inhibited by present NaF.

Effect of oxidative enzymes on bulk rheological properties of wheat flour doughs

NARCIS (Netherlands)

Dunnewind, B.; Vliet, T. van; Orsel, R.

2002-01-01

The use of enzymes such as peroxidases or glucose oxidase instead of chemical oxidants is a very interesting option for improving breadmaking performance of doughs. In this study the effect of such enzymes on bulk rheological properties of dough was quantified and their influence on the polymer

Effect of Oxidative Enzymes on Bulk Rheological Properties of Wheat Flour Doughs

NARCIS (Netherlands)

Dunnewind, B.; Vliet, van T.; Orsel, R.

2002-01-01

The use of enzymes such as peroxidases or glucose oxidase instead of chemical oxidants is a very interesting option for improving breadmaking performance of doughs. In this study the effect of such enzymes on bulk rheological properties of dough was quantified and their influence on the polymer

Effects of curcumin on angiotensin-converting enzyme gene expression, oxidative stress and anti-oxidant status in thioacetamide-induced hepatotoxicity.

Science.gov (United States)

Fazal, Yumna; Fatima, Syeda Nuzhat; Shahid, Syed Muhammad; Mahboob, Tabassum

2015-12-01

This study aimed to evaluate the protective effects of curcumin on angiotensin-converting enzyme (ACE) gene expression, oxidative stress and anti-oxidant status in thioacetamide (TAA)-induced hepatotoxicity in rats. Total 32 albino Wistar rats (male, 200-250 g) were divided into six groups (n=8). Group 1: untreated controls; Group 2: received TAA (200 mg/kg body weight (b.w.); i.p.) for 12 weeks; Group 3: received curcumin (75 mg/kg b.w.) for 24 weeks; Group 4: received TAA (200 mg/kg b.w.; i.p.) for 12 weeks+curcumin (75 mg/kg b.w.) for 12 weeks. A significantly higher ACE gene expression was observed in TAA-induced groups as compared with control, indicating more synthesis of ACE proteins. Treatment with curcumin suppressed ACE expression in TAA liver and reversed the toxicity produced. TAA treatment results in higher lipid peroxidation and lower GSH, SOD and CAT than the normal, and this produces oxidative stress in the liver. Cirrhotic conditions were confirmed by serum enzymes (ALT, AST and ALP) as well as histopathological observations. Curcumin treatment reduced oxidative stress in animals by scavenging reactive oxygen species, protecting the anti-oxidant enzymes from being denatured and reducing the oxidative stress marker lipid peroxidation. Curcumin treatment restores hepatocytes, damaged by TAA, and protects liver tissue approaching cirrhosis. © The Author(s) 2014.

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

DEFF Research Database (Denmark)

Biondi, R M; Walz, K; Issinger, O G

1996-01-01

of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

Serum prolidase enzyme activity in obese subjects and its relationship with oxidative stress markers.

Science.gov (United States)

Aslan, Mehmet; Duzenli, Ufuk; Esen, Ramazan; Soyoral, Yasemin Usul

2017-10-01

The relationship between increased serum enzyme activity of prolidase and increased rate of collagen turnover in the arterial wall has been asserted in previous studies. Collagen reflects much of the strength to the connective tissue involved in the arterial wall. Atherosclerosis is very common vessel disease and oxidative stress plays a pivotal role in the etiopathogenesis. Our objective was to examine the serum enzyme activity of prolidase and its possible relationships with oxidative stress parameters in obese subjects. Our present study was conducted 27 obese subjects and 26 age-matched healthy control subjects. The serum enzyme activity of prolidase in all study population was evaluated spectrophotometrically. Oxidative stress levels in obese subjects were analyzed with total antioxidant capacity (TAC) and total oxidant status (TOS) as well as oxidative stress index (OSI). Obese subjects have higher serum TOS and OSI indicators as well as prolidase activity than those in control subjects (for all; pstress levels in obese subjects. The significantly correlation between increased oxidative stress and increased prolidase activity may play a pivotal role in etiopathogenesis of atherosclerotic cardiovascular diseases in obese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin.

Science.gov (United States)

Rodrigues, Antônio M; Almeida, Antônio-Carlos G; Infantosi, Antonio F C; Teixeira, Hewerson Z; Duarte, Mario A

2008-02-01

The ATP hydrolysis reactions responsible for the Na(+)/K(+)-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX-Na(+)/K(+) pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX-Na(+)/K(+) pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX-Na(+)/K(+) pump complex, with similar structure to the Albers-Post model, to contribute to elucidate the PTX effect over Na(+)/K(+)-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na(+)/K(+) pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na(+)/K(+)-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX-pump complex substates for different levels of intracellular ATP concentrations.

The thermodynamic-buffer enzymes.

Science.gov (United States)

Stucki, J W

1980-08-01

Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = square root 1-q2 is fulfilled. In this relation L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load, i.e. the irreversible ATP-utilizing processes in the cell, and q the degree of coupling of oxidative phosphorylation driven by respiration. Since during short time intervals L11 and q are constant whereas L33 fluctuates in the cell, oxidative phosphorylation would only rarely operate at optimal efficiency due to violation of conductance matching. This paper demonstrates that the reversible ATP-utilizing reaction catalyzed by adenylate kinase can effectively compensate deviations from conductance matching in the presence of a fluctuating L33 and hence allows oxidative phosphorylation to operate at optimal efficiency in the cell. Since the adenylate kinase reaction was found to buffer a thermodynamic potential, i.e. the phosphate potential, this finding was generalized to the concept of thermodynamic buffering. The thermodynamic buffering ability of the adenylate kinase reaction was demonstrated by experiments with incubated rat-liver mitochondria. Considerations of changes introduced in the entropy production by the adenylate kinase reaction allowed to establish the theoretical framework for thermodynamic buffering. The ability of thermodynamic buffering to compensate deviations from conductance matching in the presence of fluctuating loads was demonstrated by computer simulations. The possibility of other reversible ATP-utilizing reactions, like the ones catalyzed by creatine kinase and arginine kinase, to contribute to thermodynamic buffering is discussed. Finally, the comparison of the theoretically calculated steady-stae cytosolic adenine nucleotide concentrations with experimental data from perfused livers demonstrated that in livers from fed rats conductance matching is fulfilled on a

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

International Nuclear Information System (INIS)

Alpert, C.A.; Frank, R.; Stueber, K.D.; Deutscher, J.; Hengstenberg, W.

1985-01-01

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate-(PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140,000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70,000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [ 32 P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group

Lipase enzymes on graphene oxide support for high-efficiency biocatalysis

Czech Academy of Sciences Publication Activity Database

Hermanová, S.; Zarevúcka, Marie; Bouša, D.; Mikulics, M.; Sofer, Z.

2016-01-01

Roč. 5, Dec (2016), s. 200-208 ISSN 2352-9407 R&D Projects: GA ČR(CZ) GA15-09001S Institutional support: RVO:61388963 Keywords : biocatalysis * graphene oxide * enzyme * acylglycerols Subject RIV: CC - Organic Chemistry

Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

DEFF Research Database (Denmark)

Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

 It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis.

Science.gov (United States)

Feichtinger, René G; Neureiter, Daniel; Skaria, Tom; Wessler, Silja; Cover, Timothy L; Mayr, Johannes A; Zimmermann, Franz A; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas ("intestinal" and "diffuse"), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

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Antonella Pantaleo

2016-01-01

Full Text Available In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.

Mitochondrial NAD(PH in vivo: identifying natural indicators of oxidative phosphorylation in the 31P magnetic resonance spectrum.

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Kevin eConley

2016-03-01

Full Text Available Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P+ and NAD(PH, which are compartmentalized between cell and mitochondria. Here we provide evidence for detection of NAD(P+ and NAD(PH in separate mitochondrial and cell pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy (31P MRS. These NAD(P pools are identified by chemical standards (NAD+, NADP+ and NADH and by physiological tests. A unique resonance reflecting mitochondrial NAD(PH is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(PH with oxidation is matched by a stoichiometric rise in the NAD(P+ peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(PH peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus non-invasive detection of NAD(P+ and NAD(PH in cell vs. mitochondria yield natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Andrographolide Attenuates LPS-Induced Cardiac Malfunctions Through Inhibition of IκB Phosphorylation and Apoptosis in Mice

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Jinlong Zhang

2015-11-01

Full Text Available Background/Aims: Cardiac malfunction is a common complication in sepsis and significantly increases the mortality of patients in septic shock. However, no studies have examined whether andrographolide (And reduces LPS-induced myocardial malfunction. Methods: Left ventricular systolic and diastolic functions were examined using echocardiography. TNF-a and IL-1ß protein levels were detected by an enzyme-linked immunosorbent assay (ELISA. NO oxidation products were determined using Griess reagent. Protein expression levels of inhibitors of NF-κBa (IκB and phospho-IκB were determined via Western blot. Oxidative injury was determined by measuring myocardial lipid peroxidation and superoxide dismutase activity. Cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL and cardiac caspase 3/7 activity. Results: And blunted LPS-induced myocardial malfunctions in mice. LPS induced TNF-a, IL-1ß, and NO production as well as I-κB phosphorylation. Cardiac apoptosis was attenuated via incubation with And, but the extent of oxidative injury remained unaffected. Conclusion: And prevents LPS-induced cardiac malfunctions in mice by inhibiting TNF-a, IL-1ß, and NO production, IκB phosphorylation, and cardiac apoptosis, indicating that And may be a potential agent for preventing myocardial malfunction during sepsis.

Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

DEFF Research Database (Denmark)

Abang Zaidel, Dayang Norulfairuz; Chronakis, Ioannis S.; Meyer, Anne S.

2013-01-01

Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w...... larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme...... catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G0) with time, were higher...

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways

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Wen Xiong

2017-09-01

Full Text Available The duck virus hepatitis (DVH caused by the duck hepatitis virus A (DHAV has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA and its phosphorylated derivative (pICA possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2 as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD and glutathione peroxidase (GSH-Px of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2 and c-Jun N-terminal kinase (JNK, which were related to mitogen-activated protein kinases (MAPKs signaling pathways. Ultimately, compared to ICA, pICA exhibited more

A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation.

Science.gov (United States)

Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

2016-01-01

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3-AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Glycogen phosphorylation and Lafora disease.

Science.gov (United States)

Roach, Peter J

2015-12-01

Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

International Nuclear Information System (INIS)

Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

1988-01-01

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

Serum Antioxidative Enzymes Levels and Oxidative Stress Products in Age-Related Cataract Patients

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Dong Chang

2013-01-01

Full Text Available Purpose. To investigate the activity of antioxidative enzymes and the products of oxidative stress in patients with age-related cataracts and compare the findings with those in healthy control subjects. Method. Sixty patients with age-related cataract and sixty healthy controls of matched age and gender were included in this study. Serum samples were obtained to detect the antioxidative enzymes of superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GSH-Px, and oxidation degradation products of malondialdehyde (MDA, 4-hydroxynonenal (4-HNE, conjugated diene (CD, advanced oxidation protein products (AOPP, protein carbonyl (PC, and 8-hydroxydeoxyguanosine (8-OHdG. Results. Serum SOD, GSH-Px, and CAT activities in cataract group were significantly decreased as compared to the control subjects (P<0.05. The levels of MDA, 4-HNE, and CD in cataract patients were significantly higher than those in the control subjects (P<0.05, P<0.01. Cataract patients had higher levels of 8-OHdG, AOPP, and PC with respect to the comparative group of normal subjects (P<0.01. And there was no statistical significance in concentration of antioxidative enzymes and oxidative stress products in patients with different subtype cataract. Conclusions. Oxidative stress is an important risk factor in the development of age-related cataract, and augmentation of the antioxidant defence systems may be of benefit to prevent or delay cataractogenesis.

The catalytic cycle of nitrous oxide reductase - The enzyme that catalyzes the last step of denitrification.

Science.gov (United States)

Carreira, Cíntia; Pauleta, Sofia R; Moura, Isabel

2017-12-01

The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper "CuZ center". This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two "CuZ center" forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic "CuZ center" is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species. Copyright © 2017 Elsevier Inc. All rights reserved.

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

International Nuclear Information System (INIS)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-01

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446 in a Nitric Oxide-Dependent Manner in C2C12 Cells

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Ruxia Wang

2018-01-01

Full Text Available Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS, the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446 and p70S6K (Thr 389. L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside, an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446/p70S6K signaling pathway in an NO-dependent manner.

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis

Science.gov (United States)

Skaria, Tom; Wessler, Silja; Cover, Timothy L.; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection. PMID:28744336

Rosamines targeting the cancer oxidative phosphorylation pathway.

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Siang Hui Lim

Full Text Available Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM, inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6 exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

OpenAIRE

Lawton, Thomas J.; Rosenzweig, Amy C.

2016-01-01

Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in tradi...

Phosphorylation states of the (Na+ + K+)-transporting ATPase in preparations from lamb kidney and electric-eel (Electophorus electricus) electric organ.

Science.gov (United States)

Harris, W E; Stahl, W L

1984-01-01

Phosphorylation states of the (Na+ + K+)-transporting ATPase were studied in highly purified preparations isolated from electric-eel electric organ and from lamb kidney. The steady-state level of phosphorylated lamb kidney enzyme, obtained by reaction with [gamma-32P]ATP, was not appreciably reduced in the presence of ADP unless oligomycin was present. The phosphorylated form of the electric-eel electric-organ enzyme was reduced by at least 95% under the same conditions, suggesting that the E1P state in the kidney enzyme is more transitory than that in electric organ. The level of phosphorylation from [32P]Pi was higher in the lamb kidney preparation than in the electric-organ preparation, and the difference in stimulation of phosphorylation by ouabain in the two preparations was striking. Ouabain increased the level of phosphorylation by 35% in the kidney preparation and 734% in the electric-organ preparation. The E2P state seems to be stabilized by ouabain in the latter preparation. These findings, as well as the different reactivities of the thiol groups to blocking reagents in these preparations, suggest that the tertiary structure in the enzyme isolated from these two sources is different. PMID:6324756

Minerals Masquerading As Enzymes: Abiotic Oxidation Of Soil Organic Matter In An Iron-Rich Humid Tropical Forest Soil

Science.gov (United States)

Hall, S. J.; Silver, W. L.

2010-12-01

Oxidative reactions play an important role in decomposing soil organic matter fractions that resist hydrolytic degradation, and fundamentally affect the cycling of recalcitrant soil carbon across ecosystems. Microbial extracellular oxidative enzymes (e.g. lignin peroxidases and laccases) have been assumed to provide a dominant role in catalyzing soil organic matter oxidation, while other potential oxidative mechanisms remain poorly explored. Here, we show that abiotic reactions mediated by the oxidation of ferrous iron (Fe(II)) could explain high potential oxidation rates in humid tropical forest soils, which often contain high concentrations of Fe(II) and experience rapid redox fluctuations between anaerobic and aerobic conditions. These abiotic reactions could provide an additional mechanism to explain high rates of decomposition in these ecosystems, despite frequent oxygen deficits. We sampled humid tropical forest soils in Puerto Rico, USA from various topographic positions, ranging from well-drained ridges to riparian valleys that experience broad fluctuations in redox potential. We measured oxidative activity by adding the model humic compound L-DOPA to soil slurries, followed by colorimetric measurements of the supernatant solution over time. Dilute hydrogen peroxide was added to a subset of slurries to measure peroxidative activity. We found that oxidative and peroxidative activity correlated positively with soil Fe(II) concentrations, counter to prevailing theory that low redox potential should suppress oxidative enzymes. Boiling or autoclaving sub-samples of soil slurries to denature any enzymes present typically increased peroxidative activity and did not eliminate oxidative activity, further suggesting the importance of an abiotic mechanism. We found substantial differences in the oxidation products of the L-DOPA substrate generated by our soil slurries in comparison with oxidation products generated by a purified enzyme (mushroom tyrosinase

Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

International Nuclear Information System (INIS)

Yang, Y.P; Randall, D.D.

1989-01-01

Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH 4 )SO 4 , glyoxylate and high concentration of MgCl 2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32 P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

Effect of enzyme and oxidative treatments on the properties of coarse wool and mohair

CSIR Research Space (South Africa)

Barkhuysen, FA

2005-09-01

Full Text Available and the current study focused on the effect of enzymes, either alone or in combination with an oxidative (chlorine) treatment, on the softness and other properties of the fibres. It was found that an enzyme treatment reduced the feltability of wool. Furthermore...

Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

Science.gov (United States)

Reinecke, D. M.; Bandurski, R. S.

1988-01-01

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation

International Nuclear Information System (INIS)

Sanga, Madhu; Younis, Islam R.; Tirumalai, Padma S.; Bland, Tina M.; Banaszewska, Monica; Konat, Gregory W.; Tracy, Timothy S.; Gannett, Peter M.; Callery, Patrick S.

2006-01-01

Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-β-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Energy Technology Data Exchange (ETDEWEB)

Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

2012-11-01

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation. �

Physical inactivity and muscle oxidative capacity in humans

DEFF Research Database (Denmark)

Gram, Martin; Dahl, Rannvá; Dela, Flemming

2014-01-01

Physical inactivity is associated with a high prevalence of type 2 diabetes and is an independent predictor of mortality. It is possible that the detrimental effects of physical inactivity are mediated through a lack of adequate muscle oxidative capacity. This short review will cover the present...... literature on the effects of different models of inactivity on muscle oxidative capacity in humans. Effects of physical inactivity include decreased mitochondrial content, decreased activity of oxidative enzymes, changes in markers of oxidative stress and a decreased expression of genes and contents...... of proteins related to oxidative phosphorylation. With such a substantial down-regulation, it is likely that a range of adenosine triphosphate (ATP)-dependent pathways such as calcium signalling, respiratory capacity and apoptosis are affected by physical inactivity. However, this has not been investigated...

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

International Nuclear Information System (INIS)

Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

1988-01-01

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [γ- 32 P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity

Protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with photosystem II

International Nuclear Information System (INIS)

Coughlan, S.J.; Hind, G.

1987-01-01

Thylakoid membranes were phosphorylated with [γ- 32 P]ATP and extracted with octyl glucoside and cholate. Among the radiolabeled phosphoproteins in the extract was a previously characterized protein kinase of 64-kDa apparent mass. The ability of this enzyme to undergo autophosphorylation in situ was used to monitor its distribution in the membrane. Fractionation studies showed that the kinase is confined to granal regions of the thylakoid, where it appears to be associated with the light-harvesting chlorophyll-protein complex of photosystem II. The kinetics of kinase autophosphorylation were investigated both in situ and in extracted, purified enzyme. In the membrane, autophosphorylation saturated within 20-30 min and was reversed with a half-time of 7-8 min upon removal of ATP or oxidative inactivation of the kinase; the accompanying dephosphorylation of light-harvesting complex was slower and kinetically complex. Fluoride (10 mM) inhibited these dephosphorylations. Autophosphorylation of the isolated kinase was independent of enzyme concentration, indicative of an intramolecular mechanism. A maximum of one serine residue per mole of kinase was esterified. Autophosphorylation was more rapid in the presence of histone IIIs, an exogenous substrate. Dephosphorylation of the isolated enzyme was not observed

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

Science.gov (United States)

Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

2016-05-16

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

Directory of Open Access Journals (Sweden)

Chuanyu Wei

Full Text Available Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage.Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated.Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4.This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the myocardium under oxidative stress.

Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

Science.gov (United States)

Hromadkova, Lenka; Kupcik, Rudolf; Vajrychova, Marie; Prikryl, Petr; Charvatova, Andrea; Jankovicova, Barbora; Ripova, Daniela; Bilkova, Zuzana; Slovakova, Marcela

2018-01-15

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni 2+ , or Co 3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology

DEFF Research Database (Denmark)

Abang Zaidel, Dayang Norulfairuz; Chronakis, Ioannis S.; Meyer, Anne S.

2012-01-01

Sugar beet pectin (SBP) is a marginally utilized co-processing product from sugar production from sugar beets. In this study, the kinetics of oxidative gelation of SBP, taking place via enzyme catalyzed cross-linking of ferulic acid moieties (FA), was studied using small angle oscillatory...

Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

Science.gov (United States)

Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

2008-07-16

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

Effects of bonny light crude oil on anti-oxidative enzymes and total ...

African Journals Online (AJOL)

Effects of bonny light crude oil on anti-oxidative enzymes and total proteins in Wistar rats. Christian E Odo, Okwesili FC Nwodo, Parker E Joshua, Chibuike S Ubani, Okon E Etim, Okechukwu PC Ugwu ...

Effect of turmeric and curcumin on oxidative stress and antioxidant enzymes in streptozotocin-induced diabetic rat.

Science.gov (United States)

Suryanarayana, Palla; Satyanarayana, Alleboena; Balakrishna, Nagalla; Kumar, Putcha Uday; Reddy, Geereddy Bhanuprakash

2007-12-01

There is increasing evidence that complications related to diabetes are associated with increased oxidative stress. Curcumin, an active principle of turmeric, has several biological properties, including antioxidant activity. The protective effect of curcumin and turmeric on streptozotocin (STZ)-induced oxidative stress in various tissues of rats was studied. Three-month-old Wistar-NIN rats were made diabetic by injecting STZ (35 mg/kg body weight) intraperitoneally and fed either only the AIN-93 diet or the AIN-93 diet containing 0.002% or 0.01% curcumin or 0.5% turmeric for a period of eight weeks. After eight weeks the levels of oxidative stress parameters and activity of antioxidant enzymes were determined in various tissues. STZ-induced hyperglycemia resulted in increased lipid peroxidation and protein carbonyls in red blood cells and other tissues and altered antioxidant enzyme activities. Interestingly, feeding curcumin and turmeric to the diabetic rats controlled oxidative stress by inhibiting the increase in TBARS and protein carbonyls and reversing altered antioxidant enzyme activities without altering the hyperglycemic state in most of the tissues. Turmeric and curcumin appear to be beneficial in preventing diabetes-induced oxidative stress in rats despite unaltered hyperglycemic status.

The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

Science.gov (United States)

Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

2011-04-01

Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

Science.gov (United States)

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies.

Science.gov (United States)

Korzeniewski, Bernard; Zoladz, Jerzy A

2002-07-01

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

Science.gov (United States)

Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

2010-04-20

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Oxidation-reduction enzymes of myocardium under ionizing radiation effect

International Nuclear Information System (INIS)

Uteshev, A.B.

1988-01-01

Tissue respiration proceses under radiation effect were investigated which allowed one to reveal slight biochemical disturbances in a cell which make up the base of functional changes of different organs and tissues and to get to know the essence of tissue respiration processes. An attempt to explain significant value of oxidation enzyme system radiosensitivity in the course of cell respiration process altogether is made when studying the state of separate links of oxidation-reduction chain. It is shown that at early periods of radiation injury activity of catalase, dehydrogenases (isocitric, α-ketoglutaric, malic, succinic acids) is suppressed, concentration of a number of cytochromes is reduced and general ferrum content is increased which is connected with conformation changes of ultrastructure of mitochondrial membranes

A Model of Oxidative Stress Management: Moderation of Carbohydrate Metabolizing Enzymes in SOD1-Null Drosophila melanogaster

Science.gov (United States)

Bernard, Kristine E.; Parkes, Tony L.; Merritt, Thomas J. S.

2011-01-01

The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP+ in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP+-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain. PMID

Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

Science.gov (United States)

Subramanian, Periasamy; Bupesh, Giridharan

2011-06-01

Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

Erection capability is potentiated by long-term sildenafil treatment: role of blood flow-induced endothelial nitric-oxide synthase phosphorylation.

Science.gov (United States)

Musicki, Biljana; Champion, Hunter C; Becker, Robyn E; Liu, Tongyun; Kramer, Melissa F; Burnett, Arthur L

2005-07-01

Despite demonstrated clinical efficacy of sildenafil for the temporary treatment of erectile dysfunction, the possibility that sildenafil used long-term durably augments erectile ability remains unclear. We investigated whether continuous long-term administration of sildenafil at clinically relevant levels to aged rats "primes" the penis for improved erectile ability and involves nitric oxide (NO) or RhoA/Rho-kinase signaling pathways. In aged, but not young rats, sildenafil prolonged erection and increased the protein expressions of phosphorylated endothelial NO synthase (eNOS) at serine-1177 and phosphorylated Akt at serine-473 in penes. Only in the young rat penis, protein expressions of phosphodiesterase-5 and phosphomyosin phosphatase target subunit 1, a marker of Rho-kinase activity, were increased by sildenafil. Sildenafil inhibited phosphodiesterase-5 activity in penes of young and aged rats coincident with assayed free plasma levels of the drug equivalent to clinically therapeutic measurements. We conclude that erectile ability can be enhanced under preconditions of erectile impairment by long-term inhibition of phosphodiesterase-5 and that the effect is mediated by Akt-dependent eNOS phosphorylation. The lack of erectile ability enhancement in young rats by long-term phosphodiesterase-5 inhibition may relate to restrained NO signaling by phosphodiesterase-5 up-regulation, lack of incremental Akt and eNOS phosphorylation, and heightened Rho-kinase signaling in the penis.

Analysis of effects of 2,2',5,5'-tetracholorobiphenyl on the flux control in oxidative phosphorylation system in rat liver mitochondria.

NARCIS (Netherlands)

Mildaziene, V.; Nauciene, Z.; Baniene, R.; Demin, O.V.; Krab, K.

2002-01-01

Modular kinetic analysis reveals that the environmental pollutant 2,2′,5,5′tetrachlorobiphenyl (2,2′,5,5′-TCB) affects a large number of steps in oxidative phosphorylation in rat liver mitochondria. 2,2′,5,5′-TCB increases membrane permeability to ions, and inhibits NADH dehydrogenase, cytochrome

Protein phosphorylation and its role in archaeal signal transduction

Science.gov (United States)

Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

2016-01-01

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

Science.gov (United States)

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-12-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

Science.gov (United States)

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Enzyme catalyzed oxidative cross-linking of feruloylated pectic polysaccharides from sugar beet

DEFF Research Database (Denmark)

Abang Zaidel, Dayang Norulfairuz

beet pulp as a potential starting material for production of pectin derived products which could help maintain the competitiveness of the sugar beet based industry. The overall objective of this study has been focusing on understanding the kinetics of enzyme catalyzed oxidative crosslinking......-linked by HRP catalysis in the presence of hydrogen peroxide (H2O2) to form ferulic acid dehydrodimers (diFAs). The composition of the substrate was analyzed by HPAEC, HPLC and MALDI-TOF, confirming the structural make up of the arabinan-oligosaccharide (Arabinose: 2.9- 3.4 mmol?g-1 DM; FA: 2.5-7.0 mg?g-1 DM......, identically composed, oil-in-water emulsion systems to study the effect of different methods of emulsion preparation on the emulsion stability in the presence of SBP and the kinetics of enzyme catalyzed oxidative gelation of SBP. The result shows that the different methods of emulsion preparation affect...

Oxidative stress and the antioxidant enzyme system in the developing brain

Directory of Open Access Journals (Sweden)

So-Yeon Shim

2013-03-01

Full Text Available Preterm infants are vulnerable to the oxidative stress due to the production of large amounts of free radicals, antioxidant system insufficiency, and immature oligodendroglial cells. Reactive oxygen species (ROS play a pivotal role in the development of periventricular leukomalacia. The three most common ROS are superoxide (O2&#8226;-, hydroxyl radical (OH&#8226;, and hydrogen peroxide (H2O2. Under normal physiological conditions, a balance is maintained between the production of ROS and the capacity of the antioxidant enzyme system. However, if this balance breaks down, ROS can exert toxic effects. Superoxide dismutase, glutathione peroxidase, and catalase are considered the classical antioxidant enzymes. A recently discovered antioxidant enzyme family, peroxiredoxin (Prdx, is also an important scavenger of free radicals. Prdx1 expression is induced at birth, whereas Prdx2 is constitutively expressed, and Prdx6 expression is consistent with the classical antioxidant enzymes. Several antioxidant substances have been studied as potential therapeutic agents; however, further preclinical and clinical studies are required before allowing clinical application.

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

Science.gov (United States)

Falk, Shaun P; Weisblum, Bernard

2013-03-01

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme III/sup mtl/ of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme II/sup mtl/ of Escherichia coli

International Nuclear Information System (INIS)

Reiche, B.; Frank, R.; Deutscher, J.; Meyer, N.; Hengstenberg, W.

1988-01-01

Enzyme III/sup mtl/ is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, the authors report the isolation of III/sup mtl/ from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of III/sup mtl/ with [ 32 P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase GLu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp-Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which they assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the III/sup mtl/ proteins was found to be 15,000. They have also determined the N-terminal sequence of both proteins. Comparison of the III/sup mtl/ peptide sequences and the C-terminal part of the enzyme II/sup mtl/ of Escherichia coli reveals considerable sequence homology, which supports the suggestion that II/sup mtl/ of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II

Assaying Oxidative Coupling Activity of CYP450 Enzymes.

Science.gov (United States)

Agarwal, Vinayak

2018-01-01

Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products. © 2018 Elsevier Inc. All rights reserved.

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the V600EBRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to V600EBRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably......, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to V...

Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

Science.gov (United States)

Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

2008-01-23

Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

DEFF Research Database (Denmark)

Guterbaum, Thomas Jeremy; Braunstein, Thomas Hartig; Fossum, A

2013-01-01

Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein end...

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

Science.gov (United States)

Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

2016-06-10

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

Study of the nature of the binding of phosphate residues in the phosphorylated form of succinyl-CoA synthetase from pigeon breast muscle

International Nuclear Information System (INIS)

Valiulina, D.S.; Skalbe, T.A.; Matveeva, L.N.

1987-01-01

The hydrolytic stability of the phosphorylated protein was investigated within a wide pH range. It was shown that the bond of the phosphate residue to protein in complex I is hydrolyzed at alkaline pH values (11.0 and 13.0). At acid pH values this bond is 50% hydrolyzed. The bond of the phosphate residue to protein in complex II is hydrolyzed at acid pH values and is stable at alkaline pH values of the medium. The phosphorylation reaction of the enzyme I, both with hydroxylamine and with diisopropyl fluorophosphate, led to 50% dephosphorylation of the protein. An analysis of an alkaline hydrolysate (3 N NaOH, 3 h, 100 0 C) of the radioactive phosphorylated enzyme II by ion exchange chromatography showed that the radioactive label of the protein is distributed in the fractions of 1-N- and 3-N-phosphohistidine, as well as 1,3-N-diphosphohistidine. The data obtained suggested that phosphate in the phosphorylated enzyme I is bound to protein, with the formation of acyl phosphate and phosphoester bonds. Phosphate in the phosphorylated enzyme II is bound to protein with the formation of a phosphoamide bond

Study of the nature of the binding of phosphate residues in the phosphorylated form of succinyl-CoA synthetase from pigeon breast muscle

Energy Technology Data Exchange (ETDEWEB)

Valiulina, D.S.; Skalbe, T.A.; Matveeva, L.N.

1987-01-10

The hydrolytic stability of the phosphorylated protein was investigated within a wide pH range. It was shown that the bond of the phosphate residue to protein in complex I is hydrolyzed at alkaline pH values (11.0 and 13.0). At acid pH values this bond is 50% hydrolyzed. The bond of the phosphate residue to protein in complex II is hydrolyzed at acid pH values and is stable at alkaline pH values of the medium. The phosphorylation reaction of the enzyme I, both with hydroxylamine and with diisopropyl fluorophosphate, led to 50% dephosphorylation of the protein. An analysis of an alkaline hydrolysate (3 N NaOH, 3 h, 100/sup 0/C) of the radioactive phosphorylated enzyme II by ion exchange chromatography showed that the radioactive label of the protein is distributed in the fractions of 1-N- and 3-N-phosphohistidine, as well as 1,3-N-diphosphohistidine. The data obtained suggested that phosphate in the phosphorylated enzyme I is bound to protein, with the formation of acyl phosphate and phosphoester bonds. Phosphate in the phosphorylated enzyme II is bound to protein with the formation of a phosphoamide bond.

A dual enzyme functionalized nanostructured thulium oxide based interface for biomedical application

Science.gov (United States)

Singh, Jay; Roychoudhury, Appan; Srivastava, Manish; Solanki, Pratima R.; Lee, Dong Won; Lee, Seung Hee; Malhotra, B. D.

2013-12-01

In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared nanorods has been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. The n-Tm2O3 nanorods are found to provide improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The structural and morphological studies of n-Tm2O3 nanorods have been carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. This interfacial platform has been used for fabrication of a total cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto a Tm2O3 nanostructured surface. The results of response studies of the fabricated ChEt-ChOx/n-Tm2O3/ITO bioelectrode show a broad linear range of 8-400 mg dL-1, detection limit of 19.78 mg (dL cm-2)-1, and high sensitivity of 0.9245 μA (mg per dL cm-2)-1 with a response time of 40 s. Further, this bioelectrode has been utilized for estimation of total cholesterol with negligible interference (3%) from analytes present in human serum samples. The utilization of this n-Tm2O3 modified electrode for enzyme-based biosensor analysis offers an efficient strategy and a novel interface for application of the rare earth metal oxide materials in the field of electrochemical sensors and bioelectronic devices.In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared

Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

International Nuclear Information System (INIS)

Pas, H.H.; Robillard, G.T.

1988-01-01

The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Verner, Zdeněk; Čermáková, P.; Škodová, Ingrid; Kováčová, B.; Lukeš, Julius; Horváth, A.

2014-01-01

Roč. 193, č. 1 (2014), s. 55-65 ISSN 0166-6851 R&D Projects: GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032; GA MŠk LH12104 Institutional support: RVO:60077344 Keywords : mitochondrion * oxidative phosphorylation * Trypanosoma * Leishmania * Phytomonas * Crithidia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

International Nuclear Information System (INIS)

Malloy, P.J.

1985-01-01

NADP + -specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with ( 32 P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32 P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32 P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32 P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32 P-labeled IDH was isolated from sonicated extracts of the cells. The 32 P-enzyme was carboxylmethylated and digested with trypsin. A single 32 P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32 P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue

Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

International Nuclear Information System (INIS)

Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

1989-01-01

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

OpenAIRE

Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

2014-01-01

The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

DEFF Research Database (Denmark)

Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

2016-01-01

ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... checkpoints, initiating DNA repair and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

Science.gov (United States)

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

O-GlcNAc modification: why so intimately associated with phosphorylation?

Directory of Open Access Journals (Sweden)

Ande Sudharsana R

2011-01-01

Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

DEFF Research Database (Denmark)

Lacour, S.; Bechet, E.; Cozzone, A.J.

2008-01-01

-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been......Background: In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY....../Principal Findings: Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc...

Palytoxin and the sodium/potassium pump—phosphorylation and potassium interaction

International Nuclear Information System (INIS)

Rodrigues, Antônio M; De Almeida, Antônio-Carlos G; Infantosi, Antonio F C

2009-01-01

We proposed a reaction model for investigating interactions between K + and the palytoxin–sodium–potassium (PTX–Na + /K + ) pump complex under conditions where enzyme phosphorylation may occur. The model is composed of (i) the Albers–Post model for Na + /K + –ATPase, describing Na + and K + pumping; (ii) the reaction model proposed for Na + /K + –ATPase interactions with its ligands (Na + , K + , ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the pump ligands. The simulations allow interpretation of the simultaneous action of Na + /K + –ATPase phosphorylation and K + on the PTX-induced channels. The results suggest that (i) phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K + occlusion rate, and (ii) K + causes channel blockage, increases the toxin dissociation rate and impedes the induced channel phosphorylation, implying reduction of the PTX toxic effect

Importance of glycolysis and oxidative phosphorylation in advanced melanoma

Directory of Open Access Journals (Sweden)

Ho Jonhan

2012-10-01

Full Text Available Abstract Serum lactate dehydrogenase (LDH is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS, we subsequently determined using tissue microarray (TMA analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

Identification of K_Ca3.1 channel as a novel regulator of Oxidative phosphorylation in a subset of pancreatic carcinoma cell lines

DEFF Research Database (Denmark)

Kovalenko, Ilya; Glasauer, Andrea; Schöckel, Laura

2016-01-01

, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well...

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the (V600E)BRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS......). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes...

Determination of the starch-phosphorylating enzyme activity in plant extracts

DEFF Research Database (Denmark)

Ritte, G.; Steup, M.; Kossmann, J.

2003-01-01

For quantification of alpha-glucan, water dikinase(GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and P-33-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP...... incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude...

Enzyme-Catalyzed Oxidation of 17β-Estradiol Using Immobilized Laccase from Trametes versicolor

Science.gov (United States)

Cardinal-Watkins, Chantale; Nicell, Jim A.

2011-01-01

Many natural and synthetic estrogens are amenable to oxidation through the catalytic action of oxidative enzymes such as the fungal laccase Trametes versicolor. This study focused on characterizing the conversion of estradiol (E2) using laccase that had been immobilized by covalent bonding onto silica beads contained in a bench-scale continuous-flow packed bed reactor. Conversion of E2 accomplished in the reactor declined when the temperature of the system was changed from room temperature to just above freezing at pH 5 as a result of a reduced rate of reaction rather than inactivation of the enzyme. Similarly, conversion increased when the system was brought to warmer temperatures. E2 conversion increased when the pH of the influent to the immobilized laccase reactor was changed from pH 7 to pH 5, but longer-term experiments showed that the enzyme is more stable at pH 7. Results also showed that the immobilized laccase maintained its activity when treating a constant supply of aqueous E2 at a low mean residence time over a 12-hour period and when treating a constant supply of aqueous E2 at a high mean residence time over a period of 9 days. PMID:21869925

Src kinase regulation by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Roskoski, Robert

2005-01-01

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

Science.gov (United States)

Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

2004-02-01

We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

Science.gov (United States)

Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

2016-01-01

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

Effect of different nutrient supply and other growth factors on the activity of the oxidizing enzymes in plants

Energy Technology Data Exchange (ETDEWEB)

Amberger, A

1960-01-01

Among the plants studied were french beans and peas; the oxidizing enzymes examined were ascorbic acid oxidase, cytochrome oxidase, phenol oxidase, peroxidase and catalase. Increasing the K dosage reduced enzyme activity and raised dry matter contents until at a very high dosage this action was reversed. Both N and P increased enzyme activity and yields. With B high enzyme activity and low dry matter content were both associated with deficiency and toxicity levels. Increasing the Fe dosage led to a rise in both dry matter content and enzyme activity, whereas F depressed yields and raised enzyme activity. Lack of water increased respiration. Light inhibited all enzyme activity.

Angiotensin-I Converting Enzyme (ACE Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora Hydrolysates

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Raheleh Ghanbari

2015-12-01

Full Text Available In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8% after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH (56.00% and ferrous ion-chelating (FIC (59.00% methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions.

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver.

Science.gov (United States)

Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir

2014-08-01

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

Science.gov (United States)

Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

1999-01-01

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

Star block-copolymers: Enzyme-inspired catalysts for oxidation of alcohols in water

KAUST Repository

Mugemana, Clement

2014-01-01

A number of fluorous amphiphilic star block-copolymers containing a tris(benzyltriazolylmethyl)amine motif have been prepared. These polymers assembled into well-defined nanostructures in water, and their mode of assembly could be controlled by changing the composition of the polymer. The polymers were used for enzyme-inspired catalysis of alcohol oxidation. This journal is © the Partner Organisations 2014.

Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

International Nuclear Information System (INIS)

Nyalendo, Carine; Sartelet, Hervé; Barrette, Stéphane; Ohta, Shigeru; Gingras, Denis; Béliveau, Richard

2009-01-01

Neuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression. Tyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome), and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices. Although there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP) and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006) or in neuroblastoma samples from patients younger than one year (P = 0.0002). In vitro experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells. Overall, these results suggest that tyrosine phosphorylated MT1-MMP

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Science.gov (United States)

Vilchèze, Catherine; Molle, Virginie; Carrère-Kremer, Séverine; Leiba, Jade; Mourey, Lionel; Shenai, Shubhada; Baronian, Grégory; Tufariello, Joann; Hartman, Travis; Veyron-Churlet, Romain; Trivelli, Xavier; Tiwari, Sangeeta; Weinrick, Brian; Alland, David; Guérardel, Yann; Jacobs, William R.; Kremer, Laurent

2014-01-01

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Catherine Vilchèze

2014-05-01

Full Text Available Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of

Magnetically responsive enzyme powders

Energy Technology Data Exchange (ETDEWEB)

Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

2015-04-15

Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

The HK2 Dependent "Warburg Effect" and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate.

Science.gov (United States)

Lis, Paweł; Dyląg, Mariusz; Niedźwiecka, Katarzyna; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2016-12-15

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

Science.gov (United States)

Nath, Sunil

2016-12-01

As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

Unravelling the Interactions between Hydrolytic and Oxidative Enzymes in Degradation of Lignocellulosic Biomass by Sporothrix carnis under Various Fermentation Conditions

Directory of Open Access Journals (Sweden)

Olusola A. Ogunyewo

2016-01-01

Full Text Available The mechanism underlying the action of lignocellulolytic enzymes in biodegradation of lignocellulosic biomass remains unclear; hence, it is crucial to investigate enzymatic interactions involved in the process. In this study, degradation of corn cob by Sporothrix carnis and involvement of lignocellulolytic enzymes in biodegradation were investigated over 240 h cultivation period. About 60% degradation of corn cob was achieved by S. carnis at the end of fermentation. The yields of hydrolytic enzymes, cellulase and xylanase, were higher than oxidative enzymes, laccase and peroxidase, over 144 h fermentation period. Maximum yields of cellulase (854.4 U/mg and xylanase (789.6 U/mg were at 96 and 144 h, respectively. Laccase and peroxidase were produced cooperatively with maximum yields of 489.06 U/mg and 585.39 U/mg at 144 h. Drastic decline in production of cellulase at 144 h (242.01 U/mg and xylanase at 192 h (192.2 U/mg indicates that they play initial roles in biodegradation of lignocellulosic biomass while laccase and peroxidase play later roles. Optimal degradation of corn cob (76.6% and production of hydrolytic and oxidative enzymes were achieved with 2.5% inoculum at pH 6.0. Results suggest synergy in interactions between the hydrolytic and oxidative enzymes which can be optimized for improved biodegradation.

Glycine Increases Insulin Sensitivity and Glutathione Biosynthesis and Protects against Oxidative Stress in a Model of Sucrose-Induced Insulin Resistance

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Mohammed El-Hafidi

2018-01-01

Full Text Available Oxidative stress and redox status play a central role in the link between insulin resistance (IR and lipotoxicity in metabolic syndrome. This mechanistic link may involve alterations in the glutathione redox state. We examined the effect of glycine supplementation to diet on glutathione biosynthesis, oxidative stress, IR, and insulin cell signaling in liver from sucrose-fed (SF rats characterized by IR and oxidative stress. Our hypothesis is that the correction of glutathione levels by glycine treatment leads to reduced oxidative stress, a mechanism associated with improved insulin signaling and IR. Glycine treatment decreases the levels of oxidative stress markers in liver from SF rats and increases the concentrations of glutathione (GSH and γ-glutamylcysteine and the amount of γ-glutamylcysteine synthetase (γ-GCS, a key enzyme of GSH biosynthesis in liver from SF rats. In liver from SF rats, glycine also decreases the insulin-induced phosphorylation of insulin receptor substrate-1 (ISR-1 in serine residue and increases the phosphorylation of insulin receptor β-subunit (IR-β in tyrosine residue. Thus, supplementing diets with glycine to correct GSH deficiency and to reduce oxidative stress provides significant metabolic benefits to SF rats by improving insulin sensitivity.

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

Science.gov (United States)

Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

2016-01-01

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

Science.gov (United States)

Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

2015-11-01

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

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Jun Sumaoka

2016-01-01

Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method

DEFF Research Database (Denmark)

Steen, Hanno; Pandey, Akhilesh; Andersen, Jens S

2002-01-01

mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine....... Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation....

Phenobarbital Meets Phosphorylation of Nuclear Receptors.

Science.gov (United States)

Negishi, Masahiko

2017-05-01

Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

Science.gov (United States)

Brouns, Stan J J; Walther, Jasper; Snijders, Ambrosius P L; van de Werken, Harmen J G; Willemen, Hanneke L D M; Worm, Petra; de Vos, Marjon G J; Andersson, Anders; Lundgren, Magnus; Mazon, Hortense F M; van den Heuvel, Robert H H; Nilsson, Peter; Salmon, Laurent; de Vos, Willem M; Wright, Phillip C; Bernander, Rolf; van der Oost, John

2006-09-15

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

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Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

2015-01-01

Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

Hyperthyroidism in the developing rat testis is associated with oxidative stress and hyperphosphorylated vimentin accumulation.

Science.gov (United States)

Zamoner, Ariane; Barreto, Kátia Padilha; Filho, Danilo Wilhelm; Sell, Fabíola; Woehl, Viviane Mara; Guma, Fátima Costa Rodrigues; Silva, Fátima Regina Mena Barreto; Pessoa-Pureur, Regina

2007-03-15

Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.

JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

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Michael Van Meter

2016-09-01

Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

Markers of oxidative stress and erythrocyte antioxidant enzyme activity in older men and women with differing physical activity.

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Rowiński, Rafał; Kozakiewicz, Mariusz; Kędziora-Kornatowska, Kornelia; Hübner-Woźniak, Elżbieta; Kędziora, Józef

2013-11-01

The aim of the present study was to examine the relationship between markers of oxidative stress and erythrocyte antioxidant enzyme activity and physical activity in older men and women. The present study included 481 participants (233 men and 248 women) in the age group 65-69 years (127 men and 125 women) and in the age group 90 years and over (106 men and 123 women). The classification of respondents by physical activity was based on answers to the question if, in the past 12 months, they engaged in any pastimes which require physical activity. The systemic oxidative stress status was assessed by measuring plasma iso-PGF2α and protein carbonyl concentration as well as erythrocyte antioxidant enzymes activity, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The concentration of plasma iso-PGF2α and protein carbonyls (CP) was lower in groups of younger men and women compared to the respective older groups. In all examined groups, physical activity resulted in decrease of these oxidative stress markers and simultaneously caused adaptive increase in the erythrocyte SOD activity. Additionally, in active younger men CAT, GPx, and GR activities were higher than in sedentary ones. In conclusion, oxidative stress increase is age-related, but physical activity can reduce oxidative stress markers and induce adaptive increase in the erythrocyte antioxidant enzyme activity, especially SOD, even in old and very old men and women. © 2013.

Ultrasensitivity in phosphorylation-dephosphorylation cycles with little substrate.

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Bruno M C Martins

Full Text Available Cellular decision-making is driven by dynamic behaviours, such as the preparations for sunrise enabled by circadian rhythms and the choice of cell fates enabled by positive feedback. Such behaviours are often built upon ultrasensitive responses where a linear change in input generates a sigmoidal change in output. Phosphorylation-dephosphorylation cycles are one means to generate ultrasensitivity. Using bioinformatics, we show that in vivo levels of kinases and phosphatases frequently exceed the levels of their corresponding substrates in budding yeast. This result is in contrast to the conditions often required by zero-order ultrasensitivity, perhaps the most well known means for how such cycles become ultrasensitive. We therefore introduce a mechanism to generate ultrasensitivity when numbers of enzymes are higher than numbers of substrates. Our model combines distributive and non-distributive actions of the enzymes with two-stage binding and concerted allosteric transitions of the substrate. We use analytical and numerical methods to calculate the Hill number of the response. For a substrate with [Formula: see text] phosphosites, we find an upper bound of the Hill number of [Formula: see text], and so even systems with a single phosphosite can be ultrasensitive. Two-stage binding, where an enzyme must first bind to a binding site on the substrate before it can access the substrate's phosphosites, allows the enzymes to sequester the substrate. Such sequestration combined with competition for each phosphosite provides an intuitive explanation for the sigmoidal shifts in levels of phosphorylated substrate. Additionally, we find cases for which the response is not monotonic, but shows instead a peak at intermediate levels of input. Given its generality, we expect the mechanism described by our model to often underlay decision-making circuits in eukaryotic cells.

Phosphorylation-dephosphorylation process as a probable mechanism for the diurnal regulatory changes of phosphoenolpyruvate carboxylase in CAM plants.

Science.gov (United States)

Brulfert, J; Vidal, J; Le Marechal, P; Gadal, P; Queiroz, O; Kluge, M; Kruger, I

1986-04-14

Day and night forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) were extracted from leaves of the CAM plants Kalanchoe daigremontiana, K. tubiflora and K. blossfeldiana previously fed with [32P] labelled phosphate solution. A one-step immunochemical purification followed by SDS polyacrylamide gel electrophoresis and autoradiography showed that, in all species, the night form of the enzyme was phosphorylated and not the day form. Limited acid hydrolysis of the night form and two-dimensional separation identified predominantly labelled phosphoserine and phosphothreonine. In vitro addition of exogenous acid phosphatase (EC 3.1.3.2) to desalted night form-containing extracts resulted within 30 min in a shift in PEPC enzymic properties similar to the in vivo changes from night to day form. It is suggested that phosphorylation-dephosphorylation of the enzyme could be the primary in vivo process which might explain the observed rhythmicity of enzymic properties.

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

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Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-08

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Manganese Superoxide Dismutase from Sphingobacterium sp. T2 as a Novel Bacterial Enzyme for Lignin Oxidation.

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Rashid, Goran M M; Taylor, Charles R; Liu, Yangqingxue; Zhang, Xiaoyang; Rea, Dean; Fülöp, Vilmos; Bugg, Timothy D H

2015-10-16

The valorization of aromatic heteropolymer lignin is an important unsolved problem in the development of a biomass-based biorefinery, for which novel high-activity biocatalysts are needed. Sequencing of the genomic DNA of lignin-degrading bacterial strain Sphingobacterium sp. T2 revealed no matches to known lignin-degrading genes. Proteomic matches for two manganese superoxide dismutase proteins were found in partially purified extracellular fractions. Recombinant MnSOD1 and MnSOD2 were both found to show high activity for oxidation of Organosolv and Kraft lignin, and lignin model compounds, generating multiple oxidation products. Structure determination revealed that the products result from aryl-Cα and Cα-Cβ bond oxidative cleavage and O-demethylation. The crystal structure of MnSOD1 was determined to 1.35 Å resolution, revealing a typical MnSOD homodimer harboring a five-coordinate trigonal bipyramidal Mn(II) center ligated by three His, one Asp, and a water/hydroxide in each active site. We propose that the lignin oxidation reactivity of these enzymes is due to the production of a hydroxyl radical, a highly reactive oxidant. This is the first demonstration that MnSOD is a microbial lignin-oxidizing enzyme.

Bacillus subtilis BY-kinase PtkA controls enzyme activity and localization of its protein substrates

DEFF Research Database (Denmark)

Jers, Carsten; Pedersen, Malene Mejer; Paspaliari, Dafni Katerina

2010-01-01

-phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity......A was dramatically altered in Delta ptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.......P>Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine...

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

DEFF Research Database (Denmark)

Rosenbaek, L L; Assentoft, M; Pedersen, N B

2012-01-01

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antib......The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho......-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and m...

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

NARCIS (Netherlands)

Veldhuis, Gertjan; Broos, Jaap; Poolman, Bert; Scheek, Ruud M.

2005-01-01

Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell.

A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

Science.gov (United States)

Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

2006-01-01

Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

The HK2 Dependent “Warburg Effect” and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate

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Paweł Lis

2016-12-01

Full Text Available This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the “Warburg” and “Crabtree” effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2, a major player in both the “Warburg effect” and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the “Warburg effect”, and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP. This small alkylating compound targets both the “Warburg effect”, i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as “multiple myeloma”. Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

Modeling nitrous oxide production and reduction in soil through explicit representation of denitrification enzyme kinetics.

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Zheng, Jianqiu; Doskey, Paul V

2015-02-17

An enzyme-explicit denitrification model with representations for pre- and de novo synthesized enzymes was developed to improve predictions of nitrous oxide (N2O) accumulations in soil and emissions from the surface. The metabolic model of denitrification is based on dual-substrate utilization and Monod growth kinetics. Enzyme synthesis/activation was incorporated into each sequential reduction step of denitrification to regulate dynamics of the denitrifier population and the active enzyme pool, which controlled the rate function. Parameterizations were developed from observations of the dynamics of N2O production and reduction in soil incubation experiments. The model successfully reproduced the dynamics of N2O and N2 accumulation in the incubations and revealed an important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Pre-synthesized denitrification enzymes contributed 20, 13, 43, and 62% of N2O that accumulated in 48 h incubations of soil collected from depths of 0-5, 5-10, 10-15, and 15-25 cm, respectively. An enzyme activity function (E) was defined to estimate the relative concentration of active enzymes and variation in response to environmental conditions. The value of E allows for activities of pre-synthesized denitrification enzymes to be differentiated from de novo synthesized enzymes. Incorporating explicit representations of denitrification enzyme kinetics into biogeochemical models is a promising approach for accurately simulating dynamics of the production and reduction of N2O in soils.

Effects of a Brussels sprouts extract on oxidative DNA damage and metabolising enzymes in rat liver

DEFF Research Database (Denmark)

Sørensen, Mette; Jensen, B.R.; Poulsen, Henrik E.

2001-01-01

and catalase activity was also assessed in the kidneys. In order to examine a possible effect of the Brussels sprouts related to oxidative stress, we measured oxidative DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and lipid peroxidation in terms of malondialdehyde (MDA) formation...... on MDA levels were found. The present results support the data obtained in several studies that consumption of cruciferous vegetables is capable of inducing various phase II enzyme systems. However, the observed increase in oxidative DNA damage raises the question of whether greatly increased ingestion...

Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

International Nuclear Information System (INIS)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-01-01

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO

Benzene Exposure Alters Expression of Enzymes Involved in Fatty Acid β-Oxidation in Male C3H/He Mice

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Rongli Sun

2016-10-01

Full Text Available Benzene is a well-known hematotoxic carcinogen that can cause leukemia and a variety of blood disorders. Our previous study indicated that benzene disturbs levels of metabolites in the fatty acid β-oxidation (FAO pathway, which is crucial for the maintenance and function of hematopoietic and leukemic cells. The present research aims to investigate the effects of benzene on changes in the expression of key enzymes in the FAO pathway in male C3H/He mice. Results showed that benzene exposure caused reduced peripheral white blood cell (WBC, red blood cell (RBC, platelet (Pit counts, and hemoglobin (Hgb concentration. Investigation of the effects of benzene on the expression of FA transport- and β-oxidation-related enzymes showed that expression of proteins Cpt1a, Crat, Acaa2, Aldh1l2, Acadvl, Crot, Echs1, and Hadha was significantly increased. The ATP levels and mitochondrial membrane potential decreased in mice exposed to benzene. Meanwhile, reactive oxygen species (ROS, hydrogen peroxide (H2O2, and malondialdehyde (MDA levels were significantly increased in the benzene group. Our results indicate that benzene induces increased expression of FA transport and β-oxidation enzymes, mitochondrial dysfunction, and oxidative stress, which may play a role in benzene-induced hematotoxicity.

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

Science.gov (United States)

Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

2016-04-01

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

Energy Technology Data Exchange (ETDEWEB)

Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

2016-04-06

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

Role of nitric oxide and antioxidant enzymes in the pathogenesis of oral cancer.

Science.gov (United States)

Patel, Jayendrakumar B; Shah, Franky D; Shukla, Shilin N; Shah, Pankaj M; Patel, Prabhudas S

2009-01-01

Oral cancer is the leading malignancy in India. Nitric oxide and antioxidant enzymes play an important role in etiology of oral cancer. Therefore, the present study evaluates nitric oxide and antioxidant enzyme levels in healthy individual without tobacco habits (NHT, N=30) and healthy individuals with tobacco habits (WHT, n=90), patients with oral precancers (OPC, n=15) and oral cancer patients (n=126). Blood samples were collected from the subjects. NO2 + NO3 (nitrite+nitrate), superoxide dismutase (SOD) and catalase levels were estimated using highly specific spectrophotometeric methods. Statistical analysis was done by SPSS statistical software version 10. Mean plasma NO2 + NO3 levels were elevated in patients with OPC and oral cancer patients as compared to the controls. Mean activities of erythrocyte SOD and catalase were higher in WHT than NHT. Erythrocyte SOD and catalase levels were higher in WHT and patients with OPC as compared to NHT. The erythrocyte SOD and catalase activities were lower in oral cancer patients than patients with OPC. The erythrocyte SOD activity was higher in advanced oral cancer than the early disease. Erythrocyte catalase activity was lower in poorly differentiated tumors than well and moderately differentiated tumors. Pearson's correlation analysis revealed that alterations in plasma NO2 + NO3 levels were negatively associated with changes in erythrocyte SOD activities. The data revealed that the alterations in antioxidant activities were associated with production of nitric oxide in oral cancer, which may have significant role in oral carcinogenesis.

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

Science.gov (United States)

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

2016-12-16

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae*

Science.gov (United States)

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M.

2016-01-01

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1–77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1–77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. PMID:27834677

Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

International Nuclear Information System (INIS)

O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

2008-01-01

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by ∼3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 (angstrom) X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state

Immunological detection of enzymes for sulfate reduction in anaerobic methane-oxidizing consortia.

Science.gov (United States)

Milucka, Jana; Widdel, Friedrich; Shima, Seigo

2013-05-01

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) at marine gas seeps is performed by archaeal-bacterial consortia that have so far not been cultivated in axenic binary or pure cultures. Knowledge about possible biochemical reactions in AOM consortia is based on metagenomic retrieval of genes related to those in archaeal methanogenesis and bacterial sulfate reduction, and identification of a few catabolic enzymes in protein extracts. Whereas the possible enzyme for methane activation (a variant of methyl-coenzyme M reductase, Mcr) was shown to be harboured by the archaea, enzymes for sulfate activation and reduction have not been localized so far. We adopted a novel approach of fluorescent immunolabelling on semi-thin (0.3-0.5 μm) cryosections to localize two enzymes of the SR pathway, adenylyl : sulfate transferase (Sat; ATP sulfurylase) and dissimilatory sulfite reductase (Dsr) in microbial consortia from Black Sea methane seeps. Both Sat and Dsr were exclusively found in an abundant microbial morphotype (c. 50% of all cells), which was tentatively identified as Desulfosarcina/Desulfococcus-related bacteria. These results show that ANME-2 archaea in the Black Sea AOM consortia did not express bacterial enzymes of the canonical sulfate reduction pathway and thus, in contrast to previous suggestions, most likely cannot perform canonical sulfate reduction. Moreover, our results show that fluorescent immunolabelling on semi-thin cryosections which to our knowledge has been so far only applied on cell tissues, is a powerful tool for intracellular protein detection in natural microbial associations. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose.

Directory of Open Access Journals (Sweden)

Bjørge Westereng

Full Text Available Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61, some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33, this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

International Nuclear Information System (INIS)

Gunter, Thomas E.; Gerstner, Brent; Lester, Tobias; Wojtovich, Andrew P.; Malecki, Jon; Swarts, Steven G.; Brookes, Paul S.; Gavin, Claire E.; Gunter, Karlene K.

2010-01-01

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn 2+ inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn 2+ inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn 2+ inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F 1 F 0 ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn 2+ inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

Science.gov (United States)

Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

2012-01-01

Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

Hydrolysis of solubilized hemicellulose derived from wet-oxidized wheat straw by a mixture of commercial fungal enzyme preparations

Energy Technology Data Exchange (ETDEWEB)

Skammelsen Schmidt, Anette; Thomsen, Alle Belinda; Woidemann, Anders [Risoe National Lab. (Denmark); Tenkanen, Maija [VTT Biotechnology and Food Research (Finland)

1998-04-01

The enzymatic hydrolysis of the solubilized hemicellulose fraction from wet-oxidized wheat straw was investigated for quantification purposes. An optimal hydrolysis depends on factors such as composition of the applied enzyme mixture and the hydrolysis conditions (enzyme loading, hydrolysis time, pH-value, and temperature). A concentrated enzyme mixture was used in this study prepared at VTT Biotechnology and Food Research, Finland, by mixing four commercial enzyme preparations. No distinctive pH-value and temperature optima were identified after a prolonged incubation of 24 hours. By reducing the hydrolysis time to 2 hours a temperature optimum was found at 50 deg. C, where a pH-value higher than 5.2 resulted in reduced activity. An enzyme-substrate-volume-ratio of 0.042, a pH-value of 5.0, and a temperature of 50 deg. C were chosen as the best hydrolysis conditions due to an improved monosaccharide yield. The hydrolysis time was chosen to be 24 hours to ensure equilibrium and total quantification. Even under the best hydrolysis conditions, the overall sugar yield from the enzymatic hydrolysis was only 85% of that of the optimal acid hydrolysis. The glucose yield were approximately the same for the two types of hydrolyses, probably due to the high cellulase activity in the VTT-enzyme mixture. For xylose and arabinose the enzymatic hydrolysis yielded only 80% of that of the acid hydrolysis. As the pentoses existed mainly as complex polymers their degradation required many different enzymes, some of which might be missing from the VTT-enzyme mixture. Furthermore, the removal of side-choins from the xylan backbone during the wet-oxidation pretreatment process might enable the hemicellulosic polymers to interact and precipitate, hence, reducing the enzymatic digestibility of the hemicellulose. (au) 8 tabs., 10 ills., 65 refs.

Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and Caucasians in the arctic winter.

Science.gov (United States)

Gnaiger, E; Boushel, R; Søndergaard, H; Munch-Andersen, T; Damsgaard, R; Hagen, C; Díez-Sánchez, C; Ara, I; Wright-Paradis, C; Schrauwen, P; Hesselink, M; Calbet, J A L; Christiansen, M; Helge, J W; Saltin, B

2015-12-01

During evolution, mitochondrial DNA haplogroups of arctic populations may have been selected for lower coupling of mitochondrial respiration to ATP production in favor of higher heat production. We show that mitochondrial coupling in skeletal muscle of traditional and westernized Inuit habituating northern Greenland is identical to Danes of western Europe haplogroups. Biochemical coupling efficiency was preserved across variations in diet, muscle fiber type, and uncoupling protein-3 content. Mitochondrial phenotype displayed plasticity in relation to lifestyle and environment. Untrained Inuit and Danes had identical capacities to oxidize fat substrate in arm muscle, which increased in Danes during the 42 days of acclimation to exercise, approaching the higher level of the Inuit hunters. A common pattern emerges of mitochondrial acclimatization and evolutionary adaptation in humans at high latitude and high altitude where economy of locomotion may be optimized by preservation of biochemical coupling efficiency at modest mitochondrial density, when submaximum performance is uncoupled from VO2max and maximum capacities of oxidative phosphorylation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

Science.gov (United States)

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

Directory of Open Access Journals (Sweden)

Jens T Stieler

Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

Enzyme-inspired functional surfactant for aerobic oxidation of activated alcohols to aldehydes in water

KAUST Repository

Chen, Batian; Bukhriakov, Konstantin; Sougrat, Rachid; Rodionov, Valentin

2015-01-01

We describe an enzyme-inspired catalytic system based on a rationally designed multifunctional amphiphile. The resulting micelles feature metal-binding sites and stable free radical moieties as well as fluorous pockets that attract and preconcentrate molecular oxygen. In the presence of copper ions, the micelles effect chemoselective aerobic alcohol oxidation under ambient conditions in water, a transformation that is challenging to achieve nonenzymatically.

Enzyme-inspired functional surfactant for aerobic oxidation of activated alcohols to aldehydes in water

KAUST Repository

Chen, Batian

2015-02-06

We describe an enzyme-inspired catalytic system based on a rationally designed multifunctional amphiphile. The resulting micelles feature metal-binding sites and stable free radical moieties as well as fluorous pockets that attract and preconcentrate molecular oxygen. In the presence of copper ions, the micelles effect chemoselective aerobic alcohol oxidation under ambient conditions in water, a transformation that is challenging to achieve nonenzymatically.

A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

Energy Technology Data Exchange (ETDEWEB)

Littrell, BobbiJo R [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

2018-04-16

The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364

Novel Tyrosine Phosphorylation Sites in Rat Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

Science.gov (United States)

Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun; Meyer, Christian; Thangiah, Geetha; Yi, Zhengping

2012-01-01

Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states. PMID:22609512

Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

Energy Technology Data Exchange (ETDEWEB)

Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

2013-07-12

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation

DEFF Research Database (Denmark)

Dzamko, Nicolas; Schertzer, Jonathan D.; Ryall, James G.

2008-01-01

The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate...... with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase...... dead (KD) AMPK alpha2. In wild-type (WT) mice, AICAR and contraction increased AMPK alpha2 and alpha1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl-CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained...

Unusual Growth Phase and Oxygen Tension Regulation of Oxidative Stress Protection Enzymes, Catalase and Superoxide Dismutase, in the Phytopathogen Xanthomonas oryzae pv. oryzae

OpenAIRE

Chamnongpol, S.; Mongkolsuk, S.; Vattanaviboon, P.; Fuangthong, M.

1995-01-01

The enzymes catalase and superoxide dismutase play major roles in protecting phytopathogenic bacteria from oxidative stress. In Xanthomonas species, these enzymes are regulated by both growth phase and oxygen tension. The highest enzyme levels were detected within 1 h of growth. Continued growth resulted in a decline of both enzyme activities. High oxygen tension was an inducing signal for both enzyme activities. An 80,000-Da monofunctional catalase and a manganese superoxide dismutase were t...

Antioxidant enzymes in Spodoptera littoralis (Boisduval): Are they enhanced to protect gut tissues during oxidative stress?

Czech Academy of Sciences Publication Activity Database

Krishnan, Natraj; Kodrík, Dalibor

2006-01-01

Roč. 52, č. 1 (2006), s. 11-20 ISSN 0022-1910 R&D Projects: GA ČR(CZ) GA522/05/0151 Institutional research plan: CEZ:AV0Z50070508 Keywords : antioxidant enzyme * oxidative stress * allelochemicals Subject RIV: CE - Biochemistry Impact factor: 2.019, year: 2006

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

Science.gov (United States)

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

ATP-consuming and ATP-generating enzymes secreted by pancreas

DEFF Research Database (Denmark)

Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

2006-01-01

-generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes...

Trace Metal Requirements for Microbial Enzymes Involved in the Production and Consumption of Methane and Nitrous Oxide

Science.gov (United States)

Glass, Jennifer B.; Orphan, Victoria J.

2011-01-01

Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH4), and nitrous oxide (N2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH4 and N2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N2O reductase, the only known enzyme capable of microbial N2O conversion to N2, have only been found in classical denitrifiers. Accumulation of N2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide

The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

Science.gov (United States)

Wongnate, Thanyaporn; Chaiyen, Pimchai

2013-07-01

Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

Science.gov (United States)

Sun, Ren; Wang, Liya

2014-10-07

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes.

NARCIS (Netherlands)

Horn, P.J.; Carruthers, L.M.; Logie, C.; Hill, D.A.; Solomon, M.J.; Wade, P.A.; Imbalzano, A.N.; Hansen, J.; Peterson, C.L.

2002-01-01

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of

Oxidative stress and redox state-regulating enzymes have prognostic relevance in diffuse large B-cell lymphoma

Directory of Open Access Journals (Sweden)

Peroja Pekka

2012-03-01

Full Text Available Abstract Background Oxidative stress and redox-regulating enzymes may have roles both in lymphomagenesis and resistance to lymphoma therapy. Previous studies from the pre-rituximab era suggest that antioxidant enzyme expression is related to prognosis in diffuse large B-cell lymphoma (DLBCL, although these results cannot be extrapolated to patient populations undergoing modern treatment modalities. In this study we assessed expression of the oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG and nitrotyrosine and the antioxidant enzymes thioredoxin (Trx, manganese superoxide dismutase (MnSOD and glutamate-cysteine ligase (GCL via immunohistochemistry in 106 patients with DLBCL. All patients were treated with CHOP-like therapy combined with rituximab. Immunostaining results were correlated with progression-free survival, disease-specific survival and traditional prognostic factors of DLBCL. Results Strong 8-OHdG immunostaining intensity was associated with extranodal involvement (p = 0.00002, a high International Prognostic Index (p = 0.002 and strong Trx (p = 0.011 and GCL (p = 0.0003 expression. Strong Trx staining intensity was associated with poor progression-free survival (p = 0.046 and poor disease-specific survival (p = 0.015. Strong GCL immunostaining intensity predicted poor progression-free survival (p = 0.049. Patients with either strong Trx or strong nitrotyrosine expression showed significantly poorer progression-free survival (p = 0.003 and disease-specific survival (p = 0.031 compared with the other patients. Conclusions The redox state-regulating enzymes GCL and Trx are promising markers in the evaluation of DLBCL prognosis in the era of modern immunochemotherapy.

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

International Nuclear Information System (INIS)

Teruel, J.A.; Inesi, G.

1988-01-01

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca 2+ -ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca 2+ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover

Regulation of cardiac C-protein phosphorylation

International Nuclear Information System (INIS)

Titus, F.L.

1985-01-01

Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System : Mechanism of Phosphoryl-Group Transfer from Phosphoenolpyruvate to HPr

NARCIS (Netherlands)

Misset, Onno; Robillard, George T.

1982-01-01

The mechanism of phosphoryl-group transfer from phosphoenolpyruvate (PEP) to HPr, catalyzed by enzyme I of the Escherichia coli PEP-dependent phosphotransferase system, has been studied in vitro. Steady-state kinetics and isotope exchange measurements revealed that this reaction cannot be described

Effects of whole-body γ-irradiation on lipid peroxidation and anti-oxidant enzymes in the liver of N-nitrosodiethylamine-treated mice

International Nuclear Information System (INIS)

Grudzinski, I.P.; Frankiewicz-Jozko, A; Gajewska, J.; Szczypka, M.; Szymanski, A.

2000-01-01

B6c3F1 mice were treated per os with either normal saline or N-nitrosodiethylamine (NDEA) (0.01, 0.1, 1.0 or 5.0 mg/kg body weight) daily for 21 days. On day 22 nd of the experiment , the animals were whole-body γ-irradiated (10 Gy) and examined at 3.5 days post-radiation exposure. Pretreatment of mice with NDEA at the lowest dosage (0.01 and 0.1 mg/kg) increased thiobarbituric acid-reactive substances (TBARS) and catalase (CAT) activity in the liver. Since the agent at the highest doses (1.0 and 5.0 mg/kg) did not have any effects on TBARS, it was associated with the selective increase of thiol (SH) groups and GSH-linked anti-oxidant enzyme activities such as glutathione peroxidase (GPX), transferase (GST) and reductase (GR). γ-irradiation decreased TBARS and increased superoxide dismutase (SOD) and GPX activity in NDEA-treated mice. Simultaneously, γ-rays did not have any effects on GST and GR enzymes, and it slightly decreased SH groups and CAT activity. Results of the present study indicate that NDEA can promote lipid peroxidation in mice liver. γ-irradiation of mice at a dose of 10 Gy modifies the activity of hepatic anti-oxidant enzymes, which in turn can lead to the reduction of NDEA-induced lipid peroxidation and/or pro-oxidant shift(s). The anti-oxidant enzymes such as SOD and GPX are suggested to be mainly involved in this process. (author)

JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

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Ling Yan

2013-01-01

Full Text Available Excessive fluoride may cause central nervous system (CNS dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS, and NADPH oxidase (NOX is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells.

JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

Science.gov (United States)

Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

2013-01-01

Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Science.gov (United States)

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-01-01

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells. PMID:26943035

Physical inactivity and muscle oxidative capacity in humans.

Science.gov (United States)

Gram, Martin; Dahl, Rannvá; Dela, Flemming

2014-01-01

Physical inactivity is associated with a high prevalence of type 2 diabetes and is an independent predictor of mortality. It is possible that the detrimental effects of physical inactivity are mediated through a lack of adequate muscle oxidative capacity. This short review will cover the present literature on the effects of different models of inactivity on muscle oxidative capacity in humans. Effects of physical inactivity include decreased mitochondrial content, decreased activity of oxidative enzymes, changes in markers of oxidative stress and a decreased expression of genes and contents of proteins related to oxidative phosphorylation. With such a substantial down-regulation, it is likely that a range of adenosine triphosphate (ATP)-dependent pathways such as calcium signalling, respiratory capacity and apoptosis are affected by physical inactivity. However, this has not been investigated in humans, and further studies are required to substantiate this hypothesis, which could expand our knowledge of the potential link between lifestyle-related diseases and muscle oxidative capacity. Furthermore, even though a large body of literature reports the effect of physical training on muscle oxidative capacity, the adaptations that occur with physical inactivity may not always be opposite to that of physical training. Thus, it is concluded that studies on the effect of physical inactivity per se on muscle oxidative capacity in functional human skeletal muscle are warranted.

Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

Science.gov (United States)

Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

2009-10-01

1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

Consensus structures of the Mo(v) sites of sulfite-oxidizing enzymes derived from variable frequency pulsed EPR spectroscopy, isotopic labelling and DFT calculations.

Science.gov (United States)

Enemark, John H

2017-10-10

Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(v) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (P i ) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

International Nuclear Information System (INIS)

Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin; Kim, Yun Gi; Shin, Jeon-Soo; Kim, Hoguen

2012-01-01

Highlights: ► Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. ► Inhibition of PKC-ζ leads to significant reduction of the secreted HMGB1. ► Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. ► Activation of PKC-ζ in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

Aging increases oxidative stress and renal expression of oxidant and antioxidant enzymes that are associated with an increased trend in systolic blood pressure.

Science.gov (United States)

Gomes, Pedro; Simão, Sónia; Silva, Elisabete; Pinto, Vanda; Amaral, João S; Afonso, Joana; Serrão, Maria Paula; Pinho, Maria João; Soares-da-Silva, Patrício

2009-01-01

The aim of this study was to investigate whether the effects of aging on oxidative stress markers and expression of major oxidant and antioxidant enzymes associate with impairment of renal function and increases in blood pressure. To explore this, we determined age-associated changes in lipid peroxidation (urinary malondialdehyde), plasma and urinary hydrogen peroxide (H(2)O(2)) levels, as well as renal H(2)O(2) production, and the expression of oxidant and antioxidant enzymes in young (13 weeks) and old (52 weeks) male Wistar Kyoto (WKY) rats. Urinary lipid peroxidation levels and H(2)O(2) production by the renal cortex and medulla of old rats were higher than their young counterparts. This was accompanied by overexpression of NADPH oxidase components Nox4 and p22(phox) in the renal cortex of old rats. Similarly, expression of superoxide dismutase (SOD) isoforms 2 and 3 and catalase were increased in the renal cortex from old rats. Renal function parameters (creatinine clearance and fractional excretion of sodium), diastolic blood pressure and heart rate were not affected by aging, although slight increases in systolic blood pressure were observed during this 52-week period. It is concluded that overexpression of renal Nox4 and p22(phox) and the increases in renal H(2)O(2) levels in aged WKY does not associate with renal functional impairment or marked increases in blood pressure. It is hypothesized that lack of oxidative stress-associated effects in aged WKY rats may result from increases in antioxidant defenses that counteract the damaging effects of H(2)O(2).

Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

Science.gov (United States)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-07-12

Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.

Ferroxitosis: A cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma

Science.gov (United States)

Lakhter, Alexander J.; Hamilton, James; Dagher, Pierre C.; Mukkamala, Suresh; Hato, Takashi; Dong, X. Charlie; Mayo, Lindsey D.; Harris, Robert A.; Shekhar, Anantha; Ivan, Mircea; Brustovetsky, Nickolay; Naidu, Samisubbu R.

2014-01-01

Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma. PMID:25587028

The relative importance of kinetic mechanisms and variable enzyme abundances for the regulation of hepatic glucose metabolism--insights from mathematical modeling.

Science.gov (United States)

Bulik, Sascha; Holzhütter, Hermann-Georg; Berndt, Nikolaus

2016-03-02

Adaptation of the cellular metabolism to varying external conditions is brought about by regulated changes in the activity of enzymes and transporters. Hormone-dependent reversible enzyme phosphorylation and concentration changes of reactants and allosteric effectors are the major types of rapid kinetic enzyme regulation, whereas on longer time scales changes in protein abundance may also become operative. Here, we used a comprehensive mathematical model of the hepatic glucose metabolism of rat hepatocytes to decipher the relative importance of different regulatory modes and their mutual interdependencies in the hepatic control of plasma glucose homeostasis. Model simulations reveal significant differences in the capability of liver metabolism to counteract variations of plasma glucose in different physiological settings (starvation, ad libitum nutrient supply, diabetes). Changes in enzyme abundances adjust the metabolic output to the anticipated physiological demand but may turn into a regulatory disadvantage if sudden unexpected changes of the external conditions occur. Allosteric and hormonal control of enzyme activities allow the liver to assume a broad range of metabolic states and may even fully reverse flux changes resulting from changes of enzyme abundances alone. Metabolic control analysis reveals that control of the hepatic glucose metabolism is mainly exerted by enzymes alone, which are differently controlled by alterations in enzyme abundance, reversible phosphorylation, and allosteric effects. In hepatic glucose metabolism, regulation of enzyme activities by changes of reactants, allosteric effects, and reversible phosphorylation is equally important as changes in protein abundance of key regulatory enzymes.

The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

Science.gov (United States)

McCully, Kilmer S

2015-01-01

The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. © 2015 by the Association of Clinical Scientists, Inc.

Myosin light chain phosphorylation is critical for adaptation to cardiac stress.

Science.gov (United States)

Warren, Sonisha A; Briggs, Laura E; Zeng, Huadong; Chuang, Joyce; Chang, Eileen I; Terada, Ryota; Li, Moyi; Swanson, Maurice S; Lecker, Stewart H; Willis, Monte S; Spinale, Francis G; Maupin-Furlowe, Julie; McMullen, Julie R; Moss, Richard L; Kasahara, Hideko

2012-11-27

Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function

DEFF Research Database (Denmark)

Ismael, Fahd O; Barrett, Tessa J; Sheipouri, Diba

2016-01-01

with the development of atherosclerosis. In this study, we examined the effect of HOCl, HOSCN and LDL pre-treated with these oxidants on the function of lysosomal enzymes responsible for protein catabolism and lipid hydrolysis in murine macrophage-like J774A.1 cells. In each case, the cells were exposed to HOCl...... or HOSCN or LDL pre-treated with these oxidants. Lysosomal cathepsin (B, L and D) and acid lipase activities were quantified, with cathepsin and LAMP-1 protein levels determined by Western blotting. Exposure of J774A.1 cells to HOCl or HOSCN resulted in a significant decrease in the activity of the Cys......-dependent cathepsins B and L, but not the Asp-dependent cathepsin D. Cathepsins B and L were also inhibited in macrophages exposed to HOSCN-modified, and to a lesser extent, HOCl-modified LDL. No change was seen in cathepsin D activity or the expression of the cathepsin proteins or lysosomal marker protein LAMP-1...

Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

International Nuclear Information System (INIS)

Xue, Peng; Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu; Sun, Guifan; Andersen, Melvin E.; Pi, Jingbo

2011-01-01

Highlights: → In 3T3-L1 adipocytes iAs 3+ decreases insulin-stimulated glucose uptake. → iAs 3+ attenuates insulin-induced phosphorylation of AKT S473. → iAs 3+ activates the cellular adaptive oxidative stress response. → iAs 3+ impairs insulin-stimulated ROS signaling. → iAs 3+ decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 μM) inorganic arsenite (iAs 3+ ) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs 3+ exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs 3+ exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in

Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

Energy Technology Data Exchange (ETDEWEB)

Xue, Peng [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China); Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Sun, Guifan [School of Public Health, China Medical University, Shenyang 110001 (China); Andersen, Melvin E. [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)

2011-04-08

Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

Science.gov (United States)

McDonald, Tanya S; Borges, Karin

2017-07-01

To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U- 13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U- 13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U- 13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei, E-mail: fchen@wayne.edu

2014-05-01

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Yun Gi [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of); Shin, Jeon-Soo [Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Hoguen, E-mail: hkyonsei@yuhs.ac [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of)

2012-07-27

Highlights: Black-Right-Pointing-Pointer Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. Black-Right-Pointing-Pointer Inhibition of PKC-{zeta} leads to significant reduction of the secreted HMGB1. Black-Right-Pointing-Pointer Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. Black-Right-Pointing-Pointer Activation of PKC-{zeta} in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-{zeta}, {lambda}, and {iota}) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-{zeta} by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-{zeta} in colon cancer tissues. Our findings suggest that PKC-{zeta} is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

Science.gov (United States)

Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

2014-05-01

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

Parameters for measurement of oxidative stress in diabetes mellitus: applicability of enzyme-linked immunosorbent assay for clinical evaluation.

Science.gov (United States)

Noiri, Eisei; Tsukahara, Hirokazu

2005-05-01

Investigations of the mechanisms involved in the onset and progression of diabetes have recently confronted the role of reactive oxygen species (ROS) and oxidative stress. Prolonged exposure to hyperglycemic conditions induces nonenzymatic glycation of protein via the so-called Maillard reaction, resulting in Schiff-base products and Amadori products that engender ROS production. These processes initiate and exacerbate micro- and macrovascular complications in diabetes. Increased oxidative stress is induced by excessive ROS production and inadequate antioxidant defenses. Recently, oxidative stress status markers have been associated directly with the severity and prognosis of diabetes. To examine oxidative stress, reliable and high-throughput methods are needed to examine large numbers of clinical samples. The emerging availability of enzyme-linked immunosorbent assay (ELISA) for oxidative stress status markers allows its application to assessment of various pathophysiologic conditions, including diabetes. This review outlines the recent achievements of ELISA application for clinical studies elucidating oxidative stress. It introduces the potential applicability of ELISA for investigating oxidative stress in diabetes.

Solubilization of low-rank coal by Trichoderma atroviride: Evidence for the involvement of hydrolytic and oxidative enzymes by using C-14-labelled lignite

Energy Technology Data Exchange (ETDEWEB)

Holker, U.; Schmiers, H.; Grosse, S.; Winkelhofer, M.; Polsakiewicz, M.; Ludwig, S.; Dohse, J.; Hofer, M. [University of Bonn, Bonn (Germany). Inst. of Botany

2002-04-01

The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth. When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity. Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization. Hence, the basic capability of T. atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with C-14-alkyl iodide. The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of C-14 radioactivity from selectively alkylated carboxylic and phenolic OH groups. T. atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases.

Enzyme oxidation of plant galactomannans yielding biomaterials with novel properties and applications, including as delivery systems.

Science.gov (United States)

Galante, Yves M; Merlini, Luca; Silvetti, Tiziana; Campia, Paola; Rossi, Bianca; Viani, Fiorenza; Brasca, Milena

2018-06-01

New biomaterials from renewable sources and the development of "functionalized biopolymers" are fields of growing industrial interest. Plant polysaccharides represent a valid alternative to traditional synthetic polymers, which are obtained from monomers of fossil, non-renewable origin. Several polysaccharides, either in their natural or chemically/biochemically modified forms, are currently employed in the biomedical, food and feed, and industrial fields, including packaging. Sustainable biochemical reactions, such as enzyme modifications of polysaccharides, open further possibilities for new product and process innovation. In the present review, we summarize the recent progress on enzyme oxidation of galactomannans (GM) from few leguminous plants (performed either with galactose oxidase or laccase) and we focus on the versatile and easily accessible laccase/TEMPO oxidative reaction. The latter causes a steep viscosity increase of GM water solutions and a transition of the gels from a viscous to an elastic form, due to formation of emiacetalic bonds and thus of internal cross-linking of the polymers. Following lyophilization of these hydrogels, stable aerogels can be obtained, which were shown to have good potential as delivery systems (DS) of actives. The active molecules tested and herewith described are polymyxin B, an antibiotic; nisin, an antimicrobial peptide; the enzymes lysozyme, protease and lipase; the mixture of the industrial microbiocides 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). The advantages of such aerogel systems and the possibilities they open for future developments, including as DS, are described.

Mitochondrial DNA (mtDNA haplogroups and serum levels of anti-oxidant enzymes in patients with osteoarthritis

Directory of Open Access Journals (Sweden)

Fernandez-Moreno Mercedes

2011-11-01

Full Text Available Abstract Background Oxidative stress play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. To prevent this, the chondrocytes possess a well-coordinated enzymatic antioxidant system. Besides, the mitochondrial DNA (mtDNA haplogroups are associated with the OA disease. Thus, the main goal of this work is to assess the incidence of the mtDNA haplogroups on serum levels of two of the main antioxidant enzymes, Manganese Superoxide Dismutase (Mn-SOD or SOD2 and catalase, and to test the suitability of these two proteins for potential OA-related biomarkers. Methods We analyzed the serum levels of SOD2 and catalase in 73 OA patients and 77 healthy controls carrying the haplogroups J, U and H, by ELISA assay. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L scoring from Grade 0 to Grade IV. Appropriate statistical analyses were performed to test the effects of clinical variables, including gender, body mass index (BMI, age, smoking status, diagnosis, haplogroups and radiologic K/L grade on serum levels of these enzymes. Results Serum levels of SOD2 appeared statistically increased in OA patients when compared with healthy controls (p Conclusions The increased levels of SOD2 in OA patients indicate an increased oxidative stress OA-related, therefore this antioxidant enzyme could be a suitable candidate biomarker for diagnosis of OA. Mitochondrial haplogroups significantly correlates with serum levels of catalase

Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

Energy Technology Data Exchange (ETDEWEB)

Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

2012-10-24

High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

Tissue specific phosphorylation of mitochondrial proteins isolated from rat liver, heart muscle, and skeletal muscle

DEFF Research Database (Denmark)

Bak, Steffen; León, Ileana R; Jensen, Ole Nørregaard

2013-01-01

-specific phosphorylation sites were identified in tissue-specific enzymes such as those encoded by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria, and CKMT2 and CPT1B in heart and skeletal muscle. Kinase prediction showed an important role for PKA and PKC in all tissues but also for proline-directed kinases......Phosphorylation of mitochondrial proteins in a variety of biological processes is increasingly being recognized and may contribute to the differences in function and energy demands observed in mitochondria from different tissues such as liver, heart, and skeletal muscle. Here, we used a combination...... of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS on isolated mitochondria to investigate the tissue-specific mitochondrial phosphoproteomes of rat liver, heart, and skeletal muscle. In total, we identified 899 phosphorylation sites in 354 different mitochondrial proteins including...

Cell age dependent variations in oxidative protective enzymes

International Nuclear Information System (INIS)

Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

1986-01-01

Activity levels of antioxidant enzymes were correlated before and after heavy-ion exposures with cellular radiosensitivity. In preliminary feasibility experiments with human T-1 cells relatively high antioxidant enzyme levels were shown in the unirradiated G 1 phase prior to the normal DNA synthetic phase. Endogenous cellular levels of three antioxidant enzymes were measured at various times in the unirradiated human T-1 cell division cycle. The enzymes measured were: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSHPX). Unlike the case in Chinese hamster V79 cells the early data with the synchronized human cell show that in very early G 1 phase (e.g., approximately 1.5 hours after mitotic selection) there are significant peaks in the levels (U/mg cell protein) of both CAT and SOD. Both enzymes show increases as the unirradiated cells progressed from mitosis into G 1 phase while the levels of GSHPX measured in duplicate samples were somewhat more variable than was the case for the other two enzymes. Studies were made in collaboration with the Armed Forces Radiobiology Research Institute

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

Studies on meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential of pigs slaughtered at different growth stages

Science.gov (United States)

Yu, Qin Ping; Feng, Ding Yuan; Xiao, Juan; Wu, Fan; He, Xiao Jun; Xia, Min Hao; Dong, Tao; Liu, Yi Hua; Tan, Hui Ze; Zou, Shi Geng; Zheng, Tao; Ou, Xian Hua; Zuo, Jian Jun

2017-01-01

Objective This experiment investigated meat color, myoglobin content, enzyme activities, and expression of genes associated with oxidative potential of pigs slaughtered at different growth stages. Methods Sixty 4-week-old Duroc×Landrace×Yorkshire pigs were assigned to 6 replicate groups, each containing 10 pigs. One pig from each group was sacrificed at day 35, 63, 98, and 161 to isolate longissimus dorsi and triceps muscles. Results Meat color scores were higher in pigs at 35 d than those at 63 d and 98 d (pMeat color scores were correlated to the proportion of oxymyoglobin (r = 0.59, pmeat color, myoglobin content, enzyme activities, and genes associated with oxidative potential varied at different stages. PMID:28728400

Mitochondrial enzymes and endoplasmic reticulum calcium stores as targets of oxidative stress in neurodegenerative diseases.

Science.gov (United States)

Gibson, Gary E; Huang, Hsueh-Meei

2004-08-01

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Science.gov (United States)

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

DEFF Research Database (Denmark)

Krintel, Christian; Mörgelin, Matthias; Logan, Derek T

2009-01-01

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well......, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have...... a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than...

Nanodiamonds as pH-switchable oxidation and reduction catalysts with enzyme-like activities for immunoassay and antioxidant applications.

Science.gov (United States)

Chen, T M; Tian, X M; Huang, L; Xiao, J; Yang, G W

2017-10-19

Nanodiamonds (NDs) have recently become a focus of interest from the viewpoints of both science and technology. Their intriguing properties make them suitable as biologically active substrates, in biosensor applications as well as diagnostic and therapeutic biomedical imaging probes. Here, we demonstrate that NDs, as oxidation and reduction catalysts, possess intrinsic enzyme mimetic properties of oxidase, peroxidase and catalase, and these behaviors can be switched by modulating the pH value. NDs not only catalyze the reduction of oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) at acidic pH, but also catalyze the dismutation decomposition of H 2 O 2 to produce O 2 at alkaline pH. It was proposed that the molecular mechanism of their peroxidase-like activity is electron-transfer acceleration, the source of which is likely derived from oxygen containing functional groups on their surface. Based on the color reaction, a nanodiamond-based enzyme linked immunosorbent assay (ELISA) was established for the detection of immunoglobulin G (IgG). Surprisingly, NDs display an excellent antioxidant activity due to the protective effect against H 2 O 2 -induced cellular oxidative damage. These findings make NDs a promising enzyme mimetic candidate and expand their applications in biocatalysis, bioassays and nano-biomedicine.

Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

Directory of Open Access Journals (Sweden)

Weckwerth Wolfram

2005-11-01

Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to

Antioxidant Enzyme Activity, Iron Content and Lipid Oxidation of Raw and Cooked Meat of Korean Native Chickens and Other Poultry

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Muhlisin

2016-05-01

Full Text Available This study was conducted to observe antioxidant enzyme activity, iron content and lipid oxidation of Korean native chickens and other poultry. The breast and thigh meat of three Korean native chicken breeds including Woorimatdak, Hyunin black and Yeonsan ogye, and three commercial poultry breeds including the broiler, White Leghorn and Pekin duck (Anasplatyrhyncos domesticus were studied. The analyses of the antioxidant enzymes activity, iron content and lipid oxidation were performed in raw and cooked samples. The activity of catalase (CAT in the thigh meat was higher than that of the breast meat of three Korean native chickens and the broiler, respectively. The activity of glutathione peroxidase (GPx in the uncooked thigh meat of three Korean native chickens was higher than that of the breasts. The breast meat of Woorimatdak and Pekin duck had higher superoxide dismutase (SOD activity than the others, while only the thigh meat of Pekin duck had the highest activity. Cooking inactivated CAT and decreased the activity of GPx and SOD. The thigh meat of Woorimatdak, White Leghorn, Yeonsan ogye and Hyunin black contained more total iron than the breast meat of those breeds. The heme-iron lost during cooking ranged from 3.2% to 14.8%. It is noted that the thigh meat had higher thiobarbituric acid reactive substances values than the breast in all chicken breeds. Though Woorimatdak showed higher antioxidant enzyme activity and lower released-iron percentage among Korean native chickens, no differences were found on lipid oxidation. We confirm that the dark meat of poultry exhibited higher antioxidant enzyme activity and contained more iron than the white meat.

Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

DEFF Research Database (Denmark)

Prats, Clara; Cadefau, Joan A; Cussó, Roser

2005-01-01

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively....... Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed...... that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ...

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer

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Kenyon Colin P

2012-03-01

Full Text Available Abstract Background The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. Results A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. Conclusions The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the

Mitochondrial oxidative enzyme activity in individual fibre types in hypo- and hyperthyroid rat skeletal muscles.

Science.gov (United States)

Johnson, M A; Turnbull, D M

1984-04-01

Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa.

Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation.

Science.gov (United States)

Nagata, Ryuhei; Fujihashi, Masahiro; Sato, Takaaki; Atomi, Haruyuki; Miki, Kunio

2015-06-09

The TK2285 protein from Thermococcus kodakarensis was recently characterized as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work, we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1d-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.

Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+

International Nuclear Information System (INIS)

Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G.

1989-01-01

The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg 2+ . In this paper, the effect of Zn 2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg 2+ . In the presence of Zn 2+ , phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn 2+ . The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn 2+ . This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus

Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells.

Science.gov (United States)

Huang, Bin; Chen, Chang-Ting; Chen, Chi-Shia; Wang, Yun-Ming; Hsieh, Hsyue-Jen; Wang, Danny Ling

2015-09-04

Laminar shear flow triggers a signaling cascade that maintains the integrity of endothelial cells (ECs). Hydrogen sulfide (H2S), a new gasotransmitter is regarded as an upstream regulator of nitric oxide (NO). Whether the H2S-generating enzymes are correlated to the enzymes involved in NO production under shear flow conditions remains unclear as yet. In the present study, the cultured ECs were subjected to a constant shear flow (12 dyn/cm(2)) in a parallel flow chamber system. We investigated the expression of three key enzymes for H2S biosynthesis, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercapto-sulfurtransferase (3-MST). Shear flow markedly increased the level of 3-MST. Shear flow enhanced the production of H2S was determined by NBD-SCN reagent that can bind to cysteine/homocystein. Exogenous treatment of NaHS that can release gaseous H2S, ECs showed an increase of phosphorylation in Akt(S473), ERK(T202/Y204) and eNOS(S1177). This indicated that H2S can trigger the NO-production signaling cascade. Silencing of CSE, CBS and 3-MST genes by siRNA separately attenuated the phosphorylation levels of Akt(S473) and eNOS(S1177) under shear flow conditions. The particular mode of shear flow increased H2S production. The interplay between H2S and NO-generating enzymes were discussed in the present study. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

International Nuclear Information System (INIS)

Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

2008-01-01

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu.

Science.gov (United States)

Castro-Roa, Daniel; Garcia-Pino, Abel; De Gieter, Steven; van Nuland, Nico A J; Loris, Remy; Zenkin, Nikolay

2013-12-01

Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

OpenAIRE

Brown, Kim M.; Lee, Louisa C.Y; Findlay, Jane E.; Day, Jonathan P.; Baillie, George S.

2012-01-01

The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kin...

Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System. Functional Asymmetry in Enzyme I Subunits Demonstrated by Reaction with 3-Bromopyruvate

NARCIS (Netherlands)

Hoeve-Duurkens, Ria ten; Robillard, George T.

1984-01-01

In the bacterial phosphoenolpyruvate-dependent sugar transport systems, enzyme I (EI) is responsible for the initial reaction step which is the transfer of the phosphoryl group from phosphoenolpyruvate to a cytoplasmic phosphocarrier protein (HPr). The inactivation of enzyme I by the substrate

Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

Science.gov (United States)

Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

2015-11-01

The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (parginase activity (pactivity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats. © Georg Thieme Verlag KG Stuttgart · New York.

Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

International Nuclear Information System (INIS)

Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

2002-01-01

Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

Cheongsangbangpung-tang ameliorated the acute inflammatory response via the inhibition of NF-κB activation and MAPK phosphorylation.

Science.gov (United States)

Kim, Seon Young; Park, Sang Mi; Hwangbo, Min; Lee, Jong Rok; Byun, Sung Hui; Ku, Sae Kwang; Cho, Il Je; Kim, Sang Chan; Jee, Seon Young; Park, Sook Jahr

2017-01-13

Cheongsangbangpung-tang (CBT) is a traditional herbal formula used in Eastern Asia to treat heat-related diseases and swellings in the skin. The present study was conducted to evaluate the anti-inflammatory effects of cheongsangbangpung-tang extract (CBTE) both in vitro and in vivo. The in vitro effects of CBTE on the lipopolysaccharide (LPS)-induced production of inflammation-related proteins were examined in RAW 264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Inflammatory cytokines and prostaglandin E 2 (PGE 2 ) were detected using the enzyme-linked immunosorbent assay (ELISA) method. Inflammation-related proteins were detected by Western blot. The effect of CBTE on acute inflammation in vivo was evaluated using carrageenan (CA)-induced paw oedema. To evaluate the anti-inflammatory effect, paw oedema volume, thickness of the dorsum and ventrum pedis skin, number of infiltrated inflammatory cells, and number of COX-2-, iNOS-immunoreactive cells were measured. In an in vitro study, CBTE inhibited the production of NO and PGE 2 and also decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) activity, interleukin (IL)-1β, IL-6 and tumuor necrosis factor-α. In LPS-activated macrophages, nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signalling is a pivotal pathway in the inflammatory process. These plausible molecular mechanisms increased the phosphorylation of I-κBα, while the activation of NF-κB and the phosphorylation of MAPK by LPS were blocked by CBTE treatment. In our in vivo study, a CA-induced acute oedematous paw inflammation rat model was used to evaluate the anti-inflammatory effect of CBTE. CBTE significantly reduced the increases in paw swelling, skin thicknesses, infiltrated inflammatory cells and iNOS-, COX-2 positive cells induced by CA injection. Based on these results, CBTE should favourably inhibit the acute inflammatory response through

Linking Alzheimer's disease to insulin resistance: the FoxO response to oxidative stress.

Science.gov (United States)

Manolopoulos, K N; Klotz, L-O; Korsten, P; Bornstein, S R; Barthel, A

2010-11-01

Oxidative stress is an important determinant not only in the pathogenesis of Alzheimer's disease (AD), but also in insulin resistance (InsRes) and diabetic complications. Forkhead box class O (FoxO) transcription factors are involved in both insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and InsRes. For example, the expression of intra- and extracellular antioxidant enzymes, such as manganese-superoxide dismutase and selenoprotein P, is regulated by FoxO proteins, as is the expression of important hepatic enzymes of gluconeogenesis. Here, we review the molecular mechanisms involved in the pathogenesis of AD and InsRes and discuss the function of FoxO proteins in these processes. Both InsRes and oxidative stress may promote the transcriptional activity of FoxO proteins, resulting in hyperglycaemia and a further increased production of reactive oxygen species (ROS). The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of β-amyloid plaques and τ protein phosphorylation. Wnt inhibition may also result in a sustained activation of FoxO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, InsRes and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. In view of their central function in this model, FoxO proteins may provide a potential molecular target for the treatment of both InsRes and AD.

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions

DEFF Research Database (Denmark)

Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E

2017-01-01

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effec...

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

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Müller Sylke

2009-05-01

Full Text Available Abstract Background Plasmodium falciparum-parasitized red blood cells (RBCs are equipped with protective antioxidant enzymes and heat shock proteins (HSPs. The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

Science.gov (United States)

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa

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Saúl Gómez-Manzo

2015-01-01

Full Text Available Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH and the aldehyde dehydrogenase (ALDH. We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6 and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

Catalytic oxidation of concentrated orange oil phase by synthetic metallic complexes biomimetic to MMO enzyme.

Science.gov (United States)

Fernandes, Ilizandra A; Esmelindro, Maria Carolina; Corazza, Marcos L; Franceschi, Elton; Treichel, Helen; de Oliveira, Debora; Frizzo, Caren D; Oliveira, J Vladimir

2010-07-01

This paper reports the catalytic oxidation of the concentrated orange oil phase using the complexes [Fe(III)(BMPP)Cl(micro-O)Fe(III)Cl(3)], [Cu(II)(BTMEA)(2)Cl]Cl and [Co(II)(BMPP)]Cl(2) biomimetic to methane monooxygenase enzyme as catalysts and hydrogen peroxide as oxidant. The reaction products of oil oxidation, mainly nootkatone, were identified by gas chromatography/mass spectrometry. A screening of catalysts was performed through a full 2(3) experimental design, varying the temperature from 30 to 70 degrees C, the catalyst concentration from 7.0 x 10(-4) to 1.5 x 10(-3) mol L(-1) and the oxidant/substrate molar ratio from 1:1 to 3:1. The results of reaction kinetics employing the most promising catalysts showed that conversions to nootkatone of up to 8% were achieved after 16 h at 70 degrees C. The results obtained in this study in terms of nootkatone production should be considered encouraging, since a real, industrially collected, raw material, instead of pure valencene, was employed in the reaction experiments, with a final content about ten times that present in the original concentrated oil.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

International Nuclear Information System (INIS)

Azim, N.; Deery, E.; Warren, M. J.; Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B.; Coker, A.; Wood, S. P.; Akhtar, M.

2014-01-01

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form

Diagnosis and epidemiology of red blood cell enzyme disorders

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Richard Van Wijk

2013-03-01

Full Text Available The red blood cell possess an active metabolic machinery that provides the cell with energy to pump ions against electrochemical gradients, to maintain its shape, to keep hemoglobin iron in the reduced (ferrous form, and to maintain enzyme and hemoglobin sulfhydryl groups. The main source of metabolic energy comes from glucose. Glucose is metabolized through the glycolytic pathway and through the hexose monophosphate shunt. Glycolysis catabolizes glucose to pyruvate and lactate, which represent the end products of glucose metabolism in the erythrocyte. Adenosine diphosphate (ADP is phosphorylated to adenosine triphosphate (ATP, and nicotinamide adenine dinucleotide (NAD+ is reduced to NADH in glycolysis. 2,3- Bisphosphoglycerate, an important regulator of the oxygen affinity of hemoglobin, is generated during glycolysis by the Rapoport-Luebering shunt. The hexose monophosphate shunt oxidizes glucose-6-phosphate, reducing NADP+ to reduced nicotinamide adenine dinucleotide phosphate (NADPH. The red cell lacks the capacity for de novo purine synthesis but has a salvage pathway that permits synthesis of purine nucleotides from purine bases...

Unraveling the evolutionary history of the phosphoryl-transfer chain of the phosphoenolpyruvate:phosphotransferase system through phylogenetic analyses and genome context

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Zúñiga Manuel

2008-05-01

Full Text Available Abstract Background The phosphoenolpyruvate phosphotransferase system (PTS plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS. Results We have analyzed the evolutionary history of the PTS phosphoryl transfer chain (PTS-ptc components in 222 complete genomes by combining phylogenetic methods and analysis of genomic context. Phylogenetic analyses alone were not conclusive for the deepest nodes but when complemented with analyses of genomic context and functional information, the main evolutionary trends of this system could be depicted. Conclusion The PTS-ptc evolved in bacteria after the divergence of early lineages such as Aquificales, Thermotogales and Thermus/Deinococcus. The subsequent evolutionary history of the PTS-ptc varied in different bacterial lineages: vertical inheritance and lineage-specific gene losses mainly explain the current situation in Actinobacteria and Firmicutes whereas horizontal gene transfer (HGT also played a major role in Proteobacteria. Most remarkably, we have identified a HGT event from Firmicutes or Fusobacteria to the last common ancestor of the Enterobacteriaceae, Pasteurellaceae, Shewanellaceae and Vibrionaceae. This transfer led to extensive changes in the metabolic and regulatory networks of these bacteria

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative*

OpenAIRE

Kashiwayama, Yoshinori; Tomohiro, Takenori; Narita, Kotomi; Suzumura, Miyuki; Glumoff, Tuomo; Hiltunen, J. Kalervo; Van Veldhoven, Paul P.; Hatanaka, Yasumaru; Imanaka, Tsuneo

2010-01-01

Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a d...

Green tea polyphenols alter lipid metabolism in the livers of broiler chickens through increased phosphorylation of AMP-activated protein kinase.

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Jinbao Huang

Full Text Available Our previous results showed that green tea polyphenols (GTPs significantly altered the expression of lipid-metabolizing genes in the liver of chickens. However, the underlying mechanism was not elucidated. In this study, we further characterized how GTPs influence AMP-activated protein kinase (AMPK in the regulation of hepatic fat metabolism. Thirty-six male chickens were fed GTPs at a daily dose of 0, 80 or 160 mg/kg of body weight for 4 weeks. The results demonstrated that oral administration of GTPs significantly reduced hepatic lipid content and abdominal fat mass, enhanced the phosphorylation levels of AMPKα and ACACA, and altered the mRNA levels and enzymatic activities of lipid-metabolizing enzymes in the liver. These results suggested that the activation of AMPK is a potential mechanism by which GTPs regulate hepatic lipid metabolism in such a way that lipid synthesis is reduced and fat oxidation is stimulated.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

Science.gov (United States)

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Nitric oxide mitigates salt stress by regulating levels of osmolytes and antioxidant enzymes in chickpea

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Parvaiz eAhmad

2016-03-01

Full Text Available This work was designed to evaluate whether external application of nitric oxide (NO in the form of its donor S-nitroso-N-acetylpenicillamine (SNAP could mitigate the deleterious effects of NaCl stress on chickpea (Cicer arietinum L. plants. SNAP (50 μM was applied to chickpea plants grown under non-saline and saline conditions (50 and 100 mM NaCl. Salt stress negatively affected growth and biomass yield, leaf relative water content (LRWC and chlorophyll content of chickpea plants. High salinity increased electrolyte leakage, carotenoid content and the levels of osmolytes (proline, glycine betaine, soluble proteins and soluble sugars, hydrogen peroxide (H2O2 and malondialdehyde (MDA, as well as the activities of antioxidant enzymes, such as superoxide dismutase (SOD, catalase (CAT, ascorbate peroxidase (APX, and glutathione reductase (GR in chickpea plants. Expression of the representative SOD, CAT and APX genes examined was also up-regulated in chickpea plants by salt stress. On the other hand, exogenous application of NO to salinized plants enhanced the growth parameters, LRWC, photosynthetic pigment production and levels of osmolytes, as well as the activities of examined antioxidant enzymes which is correlated with up-regulation of the examined SOD, CAT and APX genes, in comparison with plants treated with NaCl only. Furthermore, electrolyte leakage, H2O2 and MDA contents showed decline in salt-stressed plants supplemented with NO as compared with those in NaCl-treated plants alone. Thus, the exogenous application of NO protected chickpea plants against salt-induced oxidative damage by enhancing the biosynthesis of antioxidant enzymes, thereby improving plant growth under saline stress. Taken together, our results demonstrate that NO has capability to mitigate the adverse effects of high salinity on chickpea plants by improving LRWC, photosynthetic pigment biosyntheses, osmolyte accumulation and antioxidative defense system.

Analysis of Protein Phosphorylation and Its Functional Impact on Protein-Protein Interactions via Text Mining of the Scientific Literature.

Science.gov (United States)

Wang, Qinghua; Ross, Karen E; Huang, Hongzhan; Ren, Jia; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

2017-01-01

Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. Their activation or inactivation dictate downstream events: what substrates are modified and their subsequent impact (e.g., activation state, localization, protein-protein interactions (PPIs)). The biomedical literature continues to be the main source of evidence for experimental information about protein phosphorylation. Automatic methods to bring together phosphorylation events and phosphorylation-dependent PPIs can help to summarize the current knowledge and to expose hidden connections. In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.

Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

Science.gov (United States)

Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

2017-01-01

ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

Science.gov (United States)

Gunasekera, Bhagya; Abou Diwan, Charbel; Altawallbeh, Ghaith; Kalil, Haitham; Maher, Shaimaa; Xu, Song; Bayachou, Mekki

2018-03-07

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Wiring of Glucose Oxidizing Flavin Adenine Dinucleotide-Dependent Enzymes by Methylene Blue-Modified Third Generation Poly(amidoamine) Dendrimers Attached to Spectroscopic Graphite Electrodes

DEFF Research Database (Denmark)

Castaing, Victor; Álvarez-Martos, Isabel; Ferapontova, Elena

2016-01-01

, characterized by the heterogeneous ET rate constant of 7.1 0.1 s1; they can be used for electronic wiring of glucose-oxidizing FAD-containing enzymes, such as hexose oxidase (HOX), and further bioelectrocatalysis of glucose oxidation, starting, at pH 7, from -100 mV vs. Ag/AgCl. Thus, dendrimer...

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

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Mohan Babu

Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

DEFF Research Database (Denmark)

Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

2016-01-01

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects...... of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50...... correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs...

Procedures and means for determining enzyme activities. Verfahren und Mittel zur Bestimmung von Enzymaktivitaeten

Energy Technology Data Exchange (ETDEWEB)

Hunger, H; Behrendt, G; Schmidt, G

1988-02-10

Aim of the invention is a procedure for the determination of phosphorylating enzyme activity by using an improved means. Cellular extracts and cell lysates, resp., are contacted with surface carriers from natural and/or synthesized polymers, containing deprotonizable groups, in presence of /sup 32/P-..gamma..-ATP and, if necessary, of ATP. The phosphorylated antibiotic is evidenced by autoradiography or any other identifying procedure. Fields of application are molecular biology, genetic engineering, biotechnology and medical special fields.

Dietary α-lactalbumin induced fatty liver by enhancing nuclear liver X receptor αβ/sterol regulatory element-binding protein-1c/PPARγ expression and minimising PPARα/carnitine palmitoyltransferase-1 expression and AMP-activated protein kinase α phosphorylation associated with atherogenic dyslipidaemia, insulin resistance and oxidative stress in Balb/c mice.

Science.gov (United States)

López-Oliva, María Elvira; Garcimartin, Alba; Muñoz-Martínez, Emilia

2017-12-01

The effect and the role played by dietary α-lactalbumin (α-LAC) on hepatic fat metabolism are yet to be fully elucidated. We reported previously that α-LAC intake induced atherogenic dyslipidaemia in Balb/c mice. The aim of the present study was to investigate if this atherogenic effect could be due to a possible α-LAC-induced hepatic steatosis. We examine the ability of dietary α-LAC to induce liver steatosis, identifying the molecular mechanisms underlying hepatic lipid metabolism in association with the lipid profile, peripheral insulin resistance (IR) and changes in the hepatic oxidative environment. Male Balb/c mice (n 6) were fed with diets containing either chow or 14 % α-LAC for 4 weeks. The α-LAC-fed mice developed abdominal adiposity and IR. Moderate liver steatosis with increased TAG and NEFA contents was correlated with atherogenic dyslipidaemia. There was increased nuclear expression of liver X receptor αβ (LXRαβ), sterol regulatory element-binding protein-1c (SREBP-1c) and PPARγ transcription factors and of the cytosolic enzymes acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase involved in the hepatic de novo lipogenesis. The opposite was found for the nuclear receptor PPARα and the mitochondrial enzyme carnitine palmitoyltransferase-1 (CPT-1), leading to reduced fatty acid β-oxidation (FAO). These changes were associated with a significant decrease in both p-Thr172-AMP-activated protein kinase α (AMPKα) (inactivation) and p-Ser79-ACC1 (activation) and with a more oxidative liver environment increasing lipid peroxidation and protein oxidation and reducing GSH:GSSG ratio in the α-LAC-fed mice. In conclusion, 4 weeks of 14 % α-LAC feeding induced liver steatosis associated with atherogenic dyslipidaemia, IR and oxidative stress by enhancing nuclear LXRαβ/SREBP-1c/PPARγ expression and diminishing PPARα/CPT-1 expression and AMPKα phosphorylation shifting the hepatic FAO toward fatty acid synthesis in Balb/c mice.

Combining Solvent Isotope Effects with Substrate Isotope Effects in Mechanistic Studies of Alcohol and Amine Oxidation by Enzymes*

Science.gov (United States)

Fitzpatrick, Paul F.

2014-01-01

Oxidation of alcohols and amines is catalyzed by multiple families of flavin-and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. PMID:25448013

The Stromal Microenvironment Modulates Mitochondrial Oxidative Phosphorylation in Chronic Lymphocytic Leukemia Cells

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Hima V. Vangapandu

2017-10-01

Full Text Available Peripheral blood chronic lymphocytic leukemia (CLL cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow–derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively. Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.

Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate

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Timothy eHackmann

2015-06-01

Full Text Available From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio spp., we have re-evaluated the contribution of electron transport phosphorylation to ATP formation in this group. This group is unique in that most (76% genomes were predicted to possess genes for both Ech and Rnf transmembrane ion pumps. These pumps act in concert with the NifJ and Bcd-Etf to form a electrochemical potential (ΔμH+ and ΔμNa+, which drives ATP synthesis by electron transport phosphorylation. Of the 62 total butyrivibrio genomes currently available from the Hungate 1000 project, all 62 were predicted to possess NifJ, which reduces oxidized ferredoxin (Fdox during pyruvate conversion to acetyl-CoA. All 62 possessed all subunits of Bcd-Etf, which reduces Fdox and oxidizes reduced NAD (NADred during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH+ or ΔμNa+ from oxidation of reduced Fd and reduction of oxidized NAD (NADox. Further, 47 genomes possessed all 6 subunits of the Ech, which generates ΔμH+ from oxidation of reduced Fd (Fdred. For glucose fermentation to butyrate and H2, the electrochemical potential established should drive synthesis of ~1.5 ATP by the F0F1-ATP synthase (possessed by all 62 genomes. The total yield is ~4.5 ATP/glucose after accounting for 3 ATP formed by classic substrate-level phosphorylation, and it is one the highest yields for any glucose fermentation. The yield was the same when unsaturated fatty acid bonds, not H+, served as the electron acceptor (as during biohydrogenation. Possession of both Ech and Rnf had been previously documented in only a few sulfate-reducers, was rare in other rumen prokaryotic genomes in our analysis, and may confer an energetic advantage to rumen butyrivibrios. This unique energy conservation system might enhance the butyrivibrios’ ability to overcome growth inhibition by unsaturated fatty acids, as postulated herein.

Green tea diet decreases PCB 126-induced oxidative stress in mice by upregulating antioxidant enzymes

Science.gov (United States)

Newsome, Bradley J; Petriello, Michael C; Han, Sung Gu; Murphy, Margaret O; Eske, Katryn E; Sunkara, Manjula; Morris, Andrew J; Hennig, Bernhard

2013-01-01

Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the upregulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 μmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-Isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited five-fold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both mRNA and protein analyses, and it was determined that many genes transcriptionally controlled by AhR and Nrf2 proteins were upregulated in PCB-exposed mice fed the green tea supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126 which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants. PMID:24378064

The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

International Nuclear Information System (INIS)

Ribeiro, Mariana P.C.; Silva, Filomena S.G.; Santos, Armanda E.; Santos, Maria S.; Custódio, José B.A.

2013-01-01

Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30 nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50 nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50 nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10 nmol/mg protein prevented and reversed the Ca 2+ -induced depolarization of ΔΨ and the release of mitochondrial Ca 2+ , similarly to cyclosporine A, indicating that EDX did not affect Ca 2+ uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma. - Highlights: ► Mitochondria are important targets of Endoxifen. ► Endoxifen prevents mitochondrial permeability transition. ► Endoxifen prevents oxidative stress in

The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

Energy Technology Data Exchange (ETDEWEB)

Ribeiro, Mariana P.C.; Silva, Filomena S.G.; Santos, Armanda E. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal); Santos, Maria S. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal); Custódio, José B.A., E-mail: custodio@ci.uc.pt [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal)

2013-02-15

Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30 nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50 nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50 nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10 nmol/mg protein prevented and reversed the Ca{sup 2+}-induced depolarization of ΔΨ and the release of mitochondrial Ca{sup 2+}, similarly to cyclosporine A, indicating that EDX did not affect Ca{sup 2+} uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma. - Highlights: ► Mitochondria are important targets of Endoxifen. ► Endoxifen prevents mitochondrial permeability transition. ► Endoxifen prevents oxidative

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

DEFF Research Database (Denmark)

Issinger, O G; Beier, H; Speichermann, N

1980-01-01

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

Science.gov (United States)

Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

2015-01-01

Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa -/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa -/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa -/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme.

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Daishi Yui

Full Text Available Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD model mice showed decreased insulin-degrading enzyme (IDE levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/- mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3; Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.

Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit.

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Igor Ruvinsky

Full Text Available BACKGROUND: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-, are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests. METHODOLOGY/PRINCIPAL FINDINGS: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/- muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG in the muscle. CONCLUSIONS/SIGNIFICANCE: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar

Positive regulation of prostate cancer cell growth by lipid droplet forming and processing enzymes DGAT1 and ABHD5.

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Mitra, Ranjana; Le, Thuc T; Gorjala, Priyatham; Goodman, Oscar B

2017-09-06

Neoplastic cells proliferate rapidly and obtain requisite building blocks by reprogramming metabolic pathways that favor growth. Previously, we observed that prostate cancer cells uptake and store lipids in the form of lipid droplets, providing building blocks for membrane synthesis, to facilitate proliferation and growth. Mechanisms of lipid uptake, lipid droplet dynamics and their contribution to cancer growth have yet to be defined. This work is focused on elucidating the prostate cancer-specific modifications in lipid storage pathways so that these modified gene products can be identified and therapeutically targeted. To identify genes that promote lipid droplet formation and storage, the expression profiles of candidate genes were assessed and compared between peripheral blood mononuclear cells and prostate cancer cells. Subsequently, differentially expressed genes were inhibited and growth assays performed to elucidate their role in the growth of the cancer cells. Cell cycle, apoptosis and autophagy assays were performed to ascertain the mechanism of growth inhibition. Our results indicate that DGAT1, ABHD5, ACAT1 and ATGL are overexpressed in prostate cancer cells compared to PBMCs and of these overexpressed genes, DGAT1 and ABHD5 aid in the growth of the prostate cancer cells. Blocking the expression of both DGAT1 and ABHD5 results in inhibition of growth, cell cycle block and cell death. DGAT1 siRNA treatment inhibits lipid droplet formation and leads to autophagy where as ABHD5 siRNA treatment promotes accumulation of lipid droplets and leads to apoptosis. Both the siRNA treatments reduce AMPK phosphorylation, a key regulator of lipid metabolism. While DGAT1 siRNA reduces phosphorylation of ACC, the rate limiting enzyme in de novo fat synthesis and triggers phosphorylation of raptor and ULK-1 inducing autophagy and cell death, ABHD5 siRNA decreases P70S6 phosphorylation, leading to PARP cleavage, apoptosis and cell death. Interestingly, DGAT-1 is involved

Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans

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Veronica Codoni

2016-10-01

Full Text Available Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules. Six modules were found preserved (P < 10−4 in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR = 8.9 × 10−11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS pathway. This pathway was also found significantly (FDR < 10−4 enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10−8 the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans.

Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes.

Science.gov (United States)

Kellogg, Dean L; McCammon, Karen M; Hinchee-Rodriguez, Kathryn S; Adamo, Martin L; Roman, Linda J

2017-09-01

Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H 2 O 2 ) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H 2 O 2 -induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H 2 O 2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H 2 O 2 -stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H 2 O 2 -activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance. Copyright © 2017. Published by Elsevier Inc.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

Respiration and phosphorylation in liver and kidney mitochondria of rats exposed to high-energy gamma and beta radiation

Energy Technology Data Exchange (ETDEWEB)

Mokhoreva, S I; Vetlugina, N S

1973-01-01

The effect of whole-body irradiation with ..gamma.. rays (radiation source /sup 60/Co) at 40 rad and ..beta.. rays (source, a linear accelerator, electron energy 25 MeV) at 43 rad on oxidative phosphorylation in liver and kidney mitochondria was studied in rats. Gamma radiation gradually slowed the esterification of phosphate and respiratory rate during the oxidation of succinate in the liver and kidney mitochondria. The decrease was largest on day 15 after irradiation. However, the P/O ratio did not decrease by more than 10 to 12 percent. Despite the oxidation of glutamate in the mitochondria, respiration, phosphate consumption, and P/O ratio scarcely changed. Irradiation with electrons slowed the rate of oxidation of succinate and glutamate in liver mitochondria within 3 to 7 days. Phosphate consumption decreased at the same time so that the P/O ratio remained unchanged. Beta irradiation had virtually no effect on liver mitochondria. There is a discussion of the mechanism of action of high-energy radiation on the phosphorylation system of the mitochondria.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

International Nuclear Information System (INIS)

Alonso, Jose Maria; Bielen, Abraham A.M.; Olthuis, Wouter; Kengen, Servé W.M.; Zuilhof, Han; Franssen, Maurice C.R.

2016-01-01

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH_2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

Energy Technology Data Exchange (ETDEWEB)

Alonso, Jose Maria; Bielen, Abraham A.M. [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Olthuis, Wouter [BIOS Lab on a Chip Group, MESA+ and MIRA Institutes, University of Twente, P.O. Box 217, 7500 AE Enschede (Netherlands); Kengen, Servé W.M. [Laboratory of Microbiology, Wageningen University, 6703HB Wageningen (Netherlands); Zuilhof, Han, E-mail: han.zuilhof@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah 22254 (Saudi Arabia); Franssen, Maurice C.R., E-mail: maurice.franssen@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands)

2016-10-15

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH{sub 2}-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity.

Science.gov (United States)

Jamin, Augusta; Wicklund, April; Wiebe, Matthew S

2014-05-01

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways

SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

DEFF Research Database (Denmark)

Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

2008-01-01

spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

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Hastie C James

2006-01-01

Full Text Available Abstract Background Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

Science.gov (United States)

Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

2010-08-01

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

Curcumin Induces Nrf2 Nuclear Translocation and Prevents Glomerular Hypertension, Hyperfiltration, Oxidant Stress, and the Decrease in Antioxidant Enzymes in 5/6 Nephrectomized Rats

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Edilia Tapia

2012-01-01

Full Text Available Renal injury resulting from renal ablation induced by 5/6 nephrectomy (5/6NX is associated with oxidant stress, glomerular hypertension, hyperfiltration, and impaired Nrf2-Keap1 pathway. The purpose of this work was to know if the bifunctional antioxidant curcumin may induce nuclear translocation of Nrf2 and prevents 5/6NX-induced oxidant stress, renal injury, decrease in antioxidant enzymes, and glomerular hypertension and hyperfiltration. Four groups of rats were studied: (1 control, (2 5/6NX, (3 5/6NX +CUR, and (4 CUR (n=8–10. Curcumin was given by gavage to NX5/6 +CUR and CUR groups (60 mg/kg/day starting seven days before surgery. Rats were studied 30 days after NX5/6 or sham surgery. Curcumin attenuated 5/6NX-induced proteinuria, systemic and glomerular hypertension, hyperfiltration, glomerular sclerosis, interstitial fibrosis, interstitial inflammation, and increase in plasma creatinine and blood urea nitrogen. This protective effect was associated with enhanced nuclear translocation of Nrf2 and with prevention of 5/6NX-induced oxidant stress and decrease in the activity of antioxidant enzymes. It is concluded that the protective effect of curcumin against 5/6NX-induced glomerular and systemic hypertension, hyperfiltration, renal dysfunction, and renal injury was associated with the nuclear translocation of Nrf2 and the prevention of both oxidant stress and the decrease of antioxidant enzymes.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

Science.gov (United States)

Green, Timothy J; Dixon, Tom J; Devic, Emilie; Adlard, Robert D; Barnes, Andrew C

2009-05-01

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This

Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

Science.gov (United States)

Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

2012-10-01

Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts.

Science.gov (United States)

Liwak-Muir, Urszula; Mamady, Hapsatou; Naas, Turaya; Wylie, Quinlan; McBride, Skye; Lines, Matthew; Michaud, Jean; Baird, Stephen D; Chakraborty, Pranesh K; Holcik, Martin

2016-06-18

SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known. We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes. Our data provides further insight into cellular pathophysiology of SIFD.

Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

Science.gov (United States)

Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

2000-01-01

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

Science.gov (United States)

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

Biochemistry of storage lesions of red cell and platelet concentrates: A continuous fight implying oxidative/nitrosative/phosphorylative stress and signaling.

Science.gov (United States)

Rinalducci, Sara; Zolla, Lello

2015-06-01

The mechanisms responsible for the reduced lifespan of transfused red blood cells (RBCs) and platelets (PLTs) are still under investigation, however one explanation refers to the detrimental biochemical changes occurring during ex vivo storage of these blood products. A myriad of historical and more recent studies has contributed to advance our understanding of storage lesion. Without any doubts, proteomics had great impact on transfusion medicine by profiling the storage-dependent changes in the total detectable protein pool of both RBCs and PLTs. This review article focuses on the role of oxidative/nitrosative stress in developing RBC and PLT storage lesions, with a special glance at its biochemistry and cross-talk with phosphorylative signal transduction. In this sense, we enlighten the potential contribution of new branches of proteomics in identifying novel points of intervention for the improvement of blood product quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

Green tea diet decreases PCB 126-induced oxidative stress in mice by up-regulating antioxidant enzymes.

Science.gov (United States)

Newsome, Bradley J; Petriello, Michael C; Han, Sung Gu; Murphy, Margaret O; Eske, Katryn E; Sunkara, Manjula; Morris, Andrew J; Hennig, Bernhard

2014-02-01

Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the up-regulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low-fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 μmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited fivefold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both messenger RNA and protein analyses, and it was determined that many genes transcriptionally controlled by aryl hydrocarbon receptor and nuclear factor (erythroid-derived 2)-like 2 proteins were up-regulated in PCB-exposed mice fed the green tea-supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126, which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants. Copyright © 2014 Elsevier Inc. All rights reserved.

Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS Production

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Abrar Ul Haq Khan

2016-01-01

Full Text Available Tumor cell metabolism is altered during leukemogenesis. Cells performing oxidative phosphorylation (OXPHOS generate reactive oxygen species (ROS through mitochondrial activity. To limit the deleterious effects of excess ROS, certain gene promoters contain antioxidant response elements (ARE, e.g. the genes NQO-1 and HO-1. ROS induces conformational changes in KEAP1 and releases NRF2, which activates AREs. We show in vitro and in vivo that OXPHOS induces, both in primary leukemic cells and cell lines, de novo expression of NQO-1 and HO-1 and also the MAPK ERK5 and decreases KEAP1 mRNA. ERK5 activates the transcription factor MEF2, which binds to the promoter of the miR-23a–27a–24-2 cluster. Newly generated miR-23a destabilizes KEAP1 mRNA by binding to its 3′UTR. Lower KEAP1 levels increase the basal expression of the NRF2-dependent genes NQO-1 and HO-1. Hence, leukemic cells performing OXPHOS, independently of de novo ROS production, generate an antioxidant response to protect themselves from ROS.

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

Science.gov (United States)

Nisr, Raid B; Affourtit, Charles

2014-02-01

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies

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Israel Sánchez-Moreno

2015-11-01

Full Text Available Dihydroxyacetone (DHA kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P; a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR followed directly (without selection by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.

A simple assay method for omega-oxidation of lauric acid by hepatic enzymes

International Nuclear Information System (INIS)

Giera, D.D.; van Lier, R.B.L.

1990-01-01

Routine assessment of hepatic ω-oxidation of fatty acids in toxicology studies requires a simpler method of enzymatic analysis than HPLC or TLC. A method depending upon selective solvent separation of 14 C-lauric acid and 14 C-11/12-hydroxy lauric acid was developed. Following enzymatic incubation and addition of 15% methanol to the acidified incubation mixtures, partitioning with an alkane solvent such as iso-octane, cyclohexane, or n-hexane separated the lauric acid substrate and ω-hydroxylated products into two immiscible phases. Approximately 98% of the substrate partitioned into the organic phase, and approximately 83% of the hydroxylated products partitioned into the aqueous phase. Subsequent quantitation of the enzymatic activity required only liquid scintillation counting of the aqueous phase. Hepatic homogenates from male rats treated with 0.01, 0.05, 0.125, and 0.25% clofibrate in the diet for 7 days had enzyme levels 1.3, 6.1, 11.1, and 15.9 times control values, respectively, when assayed by conventional TLC methods, and 1.3, 5.3, 12.3, and 15.3 times control values when assayed by the solvent extraction method. The data indicate that the selective solvent partitioning yields comparable precision and sensitivity to the more conventional TLC method when studying induction of hepatic microsomal enzymes

Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

DEFF Research Database (Denmark)

Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen

2011-01-01

. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins...... in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates....... Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes....

Arsenate uncoupling of oxidative phosphorylation in isolated plant mitochondria

Energy Technology Data Exchange (ETDEWEB)

Wickes, W A; Wiskich, J T

1976-01-01

The uncoupling by arsenate of beetroot and cauliflower bud mitochondria showed the following characteristics: arsenate stimulation of respiration above the rate found with phosphate; inhibition of arsenate-stimulated respiration by phosphate; enhancement of arsenate-stimulated respiration by ADP; only partial prevention of this ADP-enhanced respiration by atractyloside; inhibition by oligomycin of the arsenate-stimulated respiration back to the phosphate rate; and the absence of any stimulatory effect of ADP in the presence of oligomycin. These results are qualitatively analogous to those reported for arsenate uncoupling in rat liver mitochondria. Arsenate stimulated malate oxidation, presumably by stimulating malate entry, in both beetroot and cauliflower bud mitochondria; however, high rates of oxidation, and presumably entry, were only sustained with arsenate in beetroot mitochondria. NADH was oxidized rapidly in cauliflower bud mitochondria in the presence of arsenate, showing that arsenate did not inhibit electron transfer processes.

Peroxiredoxin II is an antioxidant enzyme that negatively regulates collagen-stimulated platelet function.

Science.gov (United States)

Jang, Ji Yong; Wang, Su Bin; Min, Ji Hyun; Chae, Yun Hee; Baek, Jin Young; Yu, Dae-Yeul; Chang, Tong-Shin

2015-05-01

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bioavailability of catechins from guaraná (Paullinia cupana) and its effect on antioxidant enzymes and other oxidative stress markers in healthy human subjects.

Science.gov (United States)

Yonekura, Lina; Martins, Carolina Aguiar; Sampaio, Geni Rodrigues; Monteiro, Marcela Piedade; César, Luiz Antônio Machado; Mioto, Bruno Mahler; Mori, Clara Satsuki; Mendes, Thaíse Maria Nogueira; Ribeiro, Marcelo Lima; Arçari, Demetrius Paiva; Torres, Elizabeth Aparecida Ferraz da Silva

2016-07-13

We assessed the effects of guaraná (Paullinia cupana) consumption on plasma catechins, erythrocyte antioxidant enzyme activity (superoxide dismutase, catalase and glutathione peroxidase) and biomarkers of oxidative stress (ex vivo LDL oxidation, plasma total antioxidant status and ORAC, and lymphocyte single cell gel electrophoresis) in healthy overweight subjects. Twelve participants completed a 15-day run-in period followed by a 15-day intervention with a daily intake of 3 g guaraná seed powder containing 90 mg (+)-catechin and 60 mg (-)-epicatechin. Blood samples were taken on the first and last day of the intervention period, fasting and 1 h post-dose. The administration of guaraná increased plasma ORAC, while reducing ex vivo LDL oxidation (only in the first study day) and hydrogen peroxide-induced DNA damage in lymphocytes, at 1 h post-dose. Plasma catechin (0.38 ± 0.12 and 0.44 ± 0.18 nmol mL(-1)), epicatechin (0.59 ± 0.18 and 0.64 ± 0.25 nmol mL(-1)) and their methylated metabolites were observed at 1 h post-dose but were almost negligible after overnight fasting. The activities of catalase (in both study days) and glutathione peroxidase (in the last intervention day) increased at 1 h post-dose. Furthermore, the activity of both enzymes remained higher than the basal levels in overnight-fasting individuals on the last intervention day, suggesting a prolonged effect of guaraná that continues even after plasma catechin clearance. In conclusion, guaraná catechins are bioavailable and contribute to reduce the oxidative stress of clinically healthy individuals, by direct antioxidant action of the absorbed phytochemicals and up-regulation of antioxidant/detoxifying enzymes.

Phosphorylation of chicken growth hormone

International Nuclear Information System (INIS)

Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

1990-01-01

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

Increased Oxidative Stress and Imbalance in Antioxidant Enzymes in the Brains of Alloxan-Induced Diabetic Rats

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Luciane B. Ceretta

2012-01-01

Full Text Available Diabetes Mellitus (DM is associated with pathological changes in the central nervous system (SNC as well as alterations in oxidative stress. Thus, the main objective of this study was to evaluate the effects of the animal model of diabetes induced by alloxan on memory and oxidative stress. Diabetes was induced in Wistar rats by using a single injection of alloxan (150 mg/kg, and fifteen days after induction, the rats memory was evaluated through the use of the object recognition task. The oxidative stress parameters and the activity of antioxidant enzymes, superoxide dismutase (SOD, and catalase (CAT were measured in the rat brain. The results showed that diabetic rats did not have alterations in their recognition memory. However, the results did show that diabetic rats had increases in the levels of superoxide in the prefrontal cortex, and in thiobarbituric acid reactive species (TBARS production in the prefrontal cortex and in the amygdala in submitochondrial particles. Also, there was an increase in protein oxidation in the hippocampus and striatum, and in TBARS oxidation in the striatum and amygdala. The SOD activity was decreased in diabetic rats in the striatum and amygdala. However, the CAT activity was increased in the hippocampus taken from diabetic rats. In conclusion, our findings illustrate that the animal model of diabetes induced by alloxan did not cause alterations in the animals’ recognition memory, but it produced oxidants and an imbalance between SOD and CAT activities, which could contribute to the pathophysiology of diabetes.

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

Science.gov (United States)

Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

2018-05-01

Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of Grinding at Modified Atmosphere or Vacuum on Browning, Antioxidant Capacities, and Oxidative Enzyme Activities of Apple.

Science.gov (United States)

Kim, Ah-Na; Lee, Kyo-Yeon; Kim, Hyun-Jin; Chun, Jiyeon; Kerr, William L; Choi, Sung-Gil

2018-01-01

This study evaluated the effects of grinding at atmospheric pressure (control), under vacuum (∼2.67 kPa), or with modified atmosphere (N 2 and CO 2 ) on the browning, antioxidant activity, phenolics, and oxidative enzyme activity of apples as a function of time. The control group was affected most, showing distinct browning and losing most of the antioxidant activity and concentrations of the main phenolic compounds. The modified atmosphere groups retained color, antioxidant activity, and phenolic compounds better than the control group. Least changes were obtained with vacuum grinding, particularly in terms of preventing enzymatic browning and oxidation of antioxidants apples. At 12 h after grinding, vacuum-ground apples retained total phenolic contents 5.32, 1.54, and 1.49 times higher than control, nitrogen gas, and carbon dioxide gas-ground samples, respectively. The oxidative enzyme activity, including that of polyphenol oxidase and peroxidase, decreased in the control and modified atmosphere group, but they were maintained in the samples ground under the vacuum. In this study, we found that grinding with modified atmosphere or vacuum conditions could effectively prevent browning as well as loss of phenolic compounds and antioxidant activity of ground apples. These results can help scientists and engineers build better grinding systems for retaining nutrient and quality factors of ground apples. In addition, these results may be useful to other fruit and vegetable industries that wish to retain fresh-like quality and nutritional value during grinding and storage. © 2017 Institute of Food Technologists®.

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

Science.gov (United States)

Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

2016-09-27

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

Science.gov (United States)

Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

2017-09-01

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Yu, Zhenhai, E-mail: tomsyu@163.com [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China); Huang, Liangqian [Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine -SJTUSM, Shanghai, 200025 (China); Qiao, Pengyun; Jiang, Aifang; Wang, Li; Yang, Tingting [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China); Tang, Shengjian; Zhang, Wei [Plastic Surgery Institute of Weifang Medical University, Weifang, Shandong, 261041 (China); Ren, Chune, E-mail: ren@wfmc.edu.cn [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China)

2016-05-13

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. These findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.

Structures and reaction pathways of the molybdenum centres of sulfite-oxidizing enzymes by pulsed EPR spectroscopy.

Science.gov (United States)

Enemark, John H; Astashkin, Andrei V; Raitsimring, Arnold M

2008-12-01

SOEs (sulfite-oxidizing enzymes) are physiologically vital and occur in all forms of life. During the catalytic cycle, the five-co-ordinate square pyramidal oxo-molybdenum active site passes through the Mo(V) state, and intimate details of the structure can be obtained from variable frequency pulsed EPR spectroscopy through the hyperfine and nuclear quadrupole interactions of nearby magnetic nuclei. By employing variable spectrometer operational frequencies, it is possible to optimize the measurement conditions for difficult quadrupolar nuclei of interest (e.g. (17)O, (33)S, (35)Cl and (37)Cl) and to simplify the interpretation of the spectra. Isotopically labelled model Mo(V) compounds provide further insight into the electronic and geometric structures and chemical reactions of the enzymes. Recently, blocked forms of SOEs having co-ordinated sulfate, the reaction product, were detected using (33)S (I=3/2) labelling. This blocking of product release is a possible contributor to fatal human sulfite oxidase deficiency in young children.

Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.

Science.gov (United States)

Harper, M E; Brand, M D

1994-08-01

Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for -2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (delta psi m) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.

Jinlida reduces insulin resistance and ameliorates liver oxidative stress in high-fat fed rats.

Science.gov (United States)

Liu, Yixuan; Song, An; Zang, Shasha; Wang, Chao; Song, Guangyao; Li, Xiaoling; Zhu, Yajun; Yu, Xian; Li, Ling; Wang, Yun; Duan, Liyuan

2015-03-13

Jinlida (JLD) is a compound preparation formulated on the basis of traditional Chinese medicine and is officially approved for the treatment of type 2 diabetes (T2DM) in China. We aimed to elucidate the mechanism of JLD treatment, in comparison to metformin treatment, on ameliorating insulin sensitivity in insulin resistant rats and to reveal its anti-oxidant properties. Rats were fed with standard or high-fat diet for 6 weeks. After 6 weeks, the high-fat fed rats were subdivided into five groups and orally fed with JLD or metformin for 8 weeks. Fasting blood glucose (FBG), fasting blood insulin, blood lipid and antioxidant enzymes were measured. Intraperitoneal glucose tolerance test (IPGTT) and hyperinsulinemic euglycemic clamp technique were carried out to measure insulin sensitivity. Gene expression of the major signaling pathway molecules that regulate glucose uptake, including insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), phosphoinositide-3-kinase (PI3K), protein kinase beta (AKT), and glucose transporter type 2 (GLUT2), were assessed by quantitative RT-PCR. The totle and phosphorylation expression of IRS-1, AKT, JNK and p38MAPK were determined by Western blot. Treatment with JLD effectively ameliorated the high-fat induced hyperglycemia, hyperinsulinemia and hyperlipidemia. Similar to metformin, the high insulin resistance in high-fat fed rats was significantly decreased by JLD treatment. JLD displayed anti-oxidant effects, coupled with up-regulation of the insulin signaling pathway. The attenuation of hepatic oxidative stress by JLD treatment was associated with reduced phosphorylation protein levels of JNK and p38MAPK. Treatment with JLD could moderate glucose and lipid metabolism as well as reduce hepatic oxidative stress, most likely through the JNK and p38MAPK pathways. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent (CNRS-UMR); (Einstein); (TAM)

2011-08-24

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis.

Science.gov (United States)

Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C; Jacobs, William R; Kremer, Laurent

2010-12-01

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development. © 2010 Blackwell Publishing Ltd.

Inhibition of existing denitrification enzyme activity by chloramphenicol

Science.gov (United States)

Brooks, M.H.; Smith, R.L.; Macalady, D.L.

1992-01-01

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

Directory of Open Access Journals (Sweden)

So Ra Yoon

2017-01-01

Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering.

Science.gov (United States)

Kim, Jae-Eung; Kim, Eui-Jin; Chen, Hui; Wu, Chang-Hao; Adams, Michael W W; Zhang, Y-H Percival

2017-11-01

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H 2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H 2 /L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H 2 -based mass and an electricity density of more than 3000Wh/kg of starch. Copyright © 2017. Published by Elsevier Inc.

Properties of phosphorylated thymidylate synthase

DEFF Research Database (Denmark)

Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

2015-01-01

by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

Tet1 is required for Rb phosphorylation during G1/S phase transition

International Nuclear Information System (INIS)

Huang, Shengsong; Zhu, Ziqi; Wang, Yiqin; Wang, Yanru; Xu, Longxia; Chen, Xuemei; Xu, Qing; Zhang, Qimin; Zhao, Xin; Yu, Yi; Wu, Denglong

2013-01-01

Highlights: •Tet1 was required for NIT3T3 proliferation. •Tet1 depletion inhibited G1-S entry. •Cyclin D1 accumulation and Rb phosphorylation was blocked by Tet1 knockdown. -- Abstract: DNA methylation plays an important role in many biological processes, including regulation of gene expression, maintenance of chromatin conformation and genomic stability. TET-family proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which indicates that these enzymes may participate in DNA demethylation. The function of TET1 has not yet been well characterized in somatic cells. Here, we show that depletion of Tet1 in NIH3T3 cells inhibits cell growth. Furthermore, Tet1 knockdown blocks cyclin D1 accumulation in G1 phase, inhibits Rb phosphorylation and consequently delays entrance to G1/S phase. Taken together, this study demonstrates that Tet1 is required for cell proliferation and that this process is mediated through the Rb pathway

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

Science.gov (United States)

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Oishi, Akira; Ohmichi, Masahide; Takahashi, Kazuhiro; Takahashi, Toshifumi; Mori-Abe, Akiko; Kawagoe, Jun; Otsu, Reiko; Mochizuki, Yoshiko; Inaba, Noriyuki; Kurachi, Hirohisa

2004-01-01

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17β estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner

Neuroprotective effects of Arctium lappa L. roots against glutamate-induced oxidative stress by inhibiting phosphorylation of p38, JNK and ERK 1/2 MAPKs in PC12 cells.

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Tian, Xing; Sui, Shuang; Huang, Jin; Bai, Jun-Peng; Ren, Tian-Shu; Zhao, Qing-Chun

2014-07-01

Many studies have shown that glutamate-induced oxidative stress can lead to neuronal cell death involved in the development of neurodegenerative diseases. In this work, protective effects of ethyl acetate extract (EAE) of Arctium lappa L. roots against glutamate-induced oxidative stress in PC12 cells were evaluated. Also, the effects of EAE on antioxidant system, mitochondrial pathway, and signal transduction pathway were explored. Pretreatment with EAE significantly increased cell viability, activities of GSH-Px and SOD, mitochondrial membrane potential and reduced LDH leakage, ROS formation, and nuclear condensation in a dose-dependent manner. Furthermore, western blot results revealed that EAE increased the Bcl-2/Bax ratio, and inhibited the up-regulation of caspase-3, release of cytochrome c, phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). Therefore, our results indicate that EAE may be a promising neuroprotective agent for the prevention and treatment of neurodegenerative diseases implicated with oxidative stress. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation.

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Auger, Christopher; Appanna, Vasu D

2015-01-01

It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens. Copyright © 2014 Elsevier B.V. All rights reserved.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

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Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

2011-01-01

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

Nitric oxide synthase-I containing cortical interneurons co-express antioxidative enzymes and anti-apoptotic Bcl-2 following focal ischemia: evidence for direct and indirect mechanisms towards their resistance to neuropathology.

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Bidmon, H J; Emde, B; Kowalski, T; Schmitt, M; Mayer, B; Kato, K; Asayama, K; Witte, O W; Zilles, K

2001-09-01

Neuronal nitric oxide-I is constitutively expressed in approximately 2% of cortical interneurons and is co-localized with gamma-amino butric acid, somatostatin or neuropeptide Y. These interneurons additionally express high amounts of glutamate receptors which mediate the glutamate-induced hyperexcitation following cerebral injury, under these conditions nitric oxide production increases contributing to a potentiation of oxidative stress. However, perilesional nitric oxide synthase-I containing neurons are known to be resistant to ischemic and excitotoxic injury. In vitro studies show that nitrosonium and nitroxyl ions inactivate N-methyl-D-aspartate receptors, resulting in neuroprotection. The question remains of how these cells are protected against their own high intracellular nitric oxide production after activation. In this study, we investigated immunocytochemically nitric oxide synthase-I containing cortical neurons in rats after unilateral, cortical photothrombosis. In this model of focal ischemia, perilesional, constitutively nitric oxide synthase-I containing neurons survived and co-expressed antioxidative enzymes, such as manganese- and copper-zinc-dependent superoxide dismutases, heme oxygenase-2 and cytosolic glutathione peroxidase. This enhanced antioxidant expression was accompanied by a strong perinuclear presence of the antiapoptotic Bcl-2 protein. No colocalization was detectable with upregulated heme oxygenase-1 in glia and the superoxide and prostaglandin G(2)-producing cyclooxygenase-2 in neurons. These results suggest that nitric oxide synthase-I containing interneurons are protected against intracellular oxidative damage and apoptosis by Bcl-2 and several potent antioxidative enzymes. Since nitric oxide synthase-I positive neurons do not express superoxide-producing enzymes such as cyclooxygenase-1, xanthine oxidase and cyclooxygenase-2 in response to injury, this may additionally contribute to their resistance by reducing their internal

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

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Yadav, N; Kumar, S; Marlowe, T; Chaudhary, A K; Kumar, R; Wang, J; O'Malley, J; Boland, P M; Jayanthi, S; Kumar, T K S; Yadava, N; Chandra, D

2015-11-05

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency

Gene expression patterns of oxidative phosphorylation complex I subunits are organized in clusters.

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Yael Garbian

Full Text Available After the radiation of eukaryotes, the NUO operon, controlling the transcription of the NADH dehydrogenase complex of the oxidative phosphorylation system (OXPHOS complex I, was broken down and genes encoding this protein complex were dispersed across the nuclear genome. Seven genes, however, were retained in the genome of the mitochondrion, the ancient symbiote of eukaryotes. This division, in combination with the three-fold increase in subunit number from bacteria (N = approximately 14 to man (N = 45, renders the transcription regulation of OXPHOS complex I a challenge. Recently bioinformatics analysis of the promoter regions of all OXPHOS genes in mammals supported patterns of co-regulation, suggesting that natural selection favored a mechanism facilitating the transcriptional regulatory control of genes encoding subunits of these large protein complexes. Here, using real time PCR of mitochondrial (mtDNA- and nuclear DNA (nDNA-encoded transcripts in a panel of 13 different human tissues, we show that the expression pattern of OXPHOS complex I genes is regulated in several clusters. Firstly, all mtDNA-encoded complex I subunits (N = 7 share a similar expression pattern, distinct from all tested nDNA-encoded subunits (N = 10. Secondly, two sub-clusters of nDNA-encoded transcripts with significantly different expression patterns were observed. Thirdly, the expression patterns of two nDNA-encoded genes, NDUFA4 and NDUFA5, notably diverged from the rest of the nDNA-encoded subunits, suggesting a certain degree of tissue specificity. Finally, the expression pattern of the mtDNA-encoded ND4L gene diverged from the rest of the tested mtDNA-encoded transcripts that are regulated by the same promoter, consistent with post-transcriptional regulation. These findings suggest, for the first time, that the regulation of complex I subunits expression in humans is complex rather than reflecting global co-regulation.

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

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Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

2013-01-01

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

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Curran, Jerry; Tang, Lifei; Roof, Steve R; Velmurugan, Sathya; Millard, Ashley; Shonts, Stephen; Wang, Honglan; Santiago, Demetrio; Ahmad, Usama; Perryman, Matthew; Bers, Donald M; Mohler, Peter J; Ziolo, Mark T; Shannon, Thomas R

2014-01-01

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is

Mcm2 phosphorylation and the response to replicative stress

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Stead Brent E

2012-05-01

Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

Energy Technology Data Exchange (ETDEWEB)

Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

2013-04-15

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

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Patel, Anant B; Lai, James C K; Chowdhury, Golam M I; Hyder, Fahmeed; Rothman, Douglas L; Shulman, Robert G; Behar, Kevin L

2014-04-08

Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

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Nkosi Thokozani C

2011-07-01

Full Text Available Abstract Background The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the

A highly sensitive electrochemical biosensor for catechol using conducting polymer reduced graphene oxide-metal oxide enzyme modified electrode.

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Sethuraman, V; Muthuraja, P; Anandha Raj, J; Manisankar, P

2016-10-15

The fabrication, characterization and analytical performances were investigated for a catechol biosensor, based on the PEDOT-rGO-Fe2O3-PPO composite modified glassy carbon (GC) electrode. The graphene oxide (GO) doped conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) was prepared through electrochemical polymerization by potential cycling. Reduction of PEDOT-GO was carried out by amperometric method. Fe2O3 nanoparticles were synthesized in ethanol by hydrothermal method. The mixture of Fe2O3, PPO and glutaraldehyde was casted on the PEDOT-rGO electrode. The surface morphology of the modified electrodes was studied by FE-SEM and AFM. Cyclic voltammetric studies of catechol on the enzyme modified electrode revealed higher reduction peak current. Determination of catechol was carried out successfully by Differential Pulse Voltammetry (DPV) technique. The fabricated biosensor investigated shows a maximum current response at pH 6.5. The catechol biosensor exhibited wide sensing linear range from 4×10(-8) to 6.20×10(-5)M, lower detection limit of 7×10(-9)M, current maxima (Imax) of 92.55µA and Michaelis-Menten (Km) constant of 30.48µM. The activation energy (Ea) of enzyme electrode is 35.93KJmol(-1) at 50°C. There is no interference from d-glucose and l-glutamic acid, ascorbic acid and o-nitrophenol. The PEDOT-rGO-Fe2O3-PPO biosensor was stable for at least 75 days when stored in a buffer at about 4°C. Copyright © 2015 Elsevier B.V. All rights reserved.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

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Nicola V. Huck

2017-12-01

Full Text Available Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Peroxisomal enzymes in the liver of rats with experimental diabetes mellitus type 2.

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Turecký, L; Kupčová, V; Uhlíková, E; Mojto, V

2014-01-01

Diabetes mellitus is relatively frequently associated with fatty liver disease. Increased oxidative stress probably plays an important role in the development of this hepatopathy. One of possible sources of reactive oxygen species in liver is peroxisomal system. There are several reports about changes of peroxisomal enzymes in experimental diabetes, mainly enzymes of fatty acid oxidation. The aim of our study was to investigate the possible changes of activities of liver peroxisomal enzymes, other than enzymes of beta-oxidation, in experimental diabetes mellitus type 2. Biochemical changes in liver of experimental animals suggest the presence of liver steatosis. The changes of serum parameters in experimental group are similar to changes in serum of patients with non-alcoholic fatty liver disease. We have shown that diabetes mellitus influenced peroxisomal enzymes by the different way. Despite of well-known induction of peroxisomal beta-oxidation, the activities of catalase, aminoacid oxidase and NADH-cytochrome b(5) reductase were not significantly changed and the activities of glycolate oxidase and NADP-isocitrate dehydrogenase were significantly decreased. The effect of diabetes on liver peroxisomes is probably due to the increased supply of fatty acids to liver in diabetic state and also due to increased oxidative stress. The changes of metabolic activity of peroxisomal compartment may participate on the development of diabetic hepatopathy.

Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

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Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

2012-11-01

Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Hepatic Flavin-Containing Monooxygenase 3 Enzyme Suppressed by Type 1 Allergy-Produced Nitric Oxide.

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Tanino, Tadatoshi; Bando, Toru; Komada, Akira; Nojiri, Yukie; Okada, Yuna; Ueda, Yukari; Sakurai, Eiichi

2017-11-01

Flavin-containing monooxygenases (FMOs) are major mammalian non-cytochrome P450 oxidative enzymes. T helper 2 cell-activated allergic diseases produce excess levels of nitric oxide (NO) that modify the functions of proteins. However, it remains unclear whether allergy-induced NO affects the pharmacokinetics of drugs metabolized by FMOs. This study investigated alterations of hepatic microsomal FMO1 and FMO3 activities in type 1 allergic mice and further examined the interaction of FMO1 and FMO3 with allergy-induced NO. Imipramine (IMP; FMO1 substrate) N- oxidation activity was not altered in allergic mice with high serum NO and immunoglobulin E levels. At 7 days after primary sensitization (PS7) or secondary sensitization (SS7), benzydamine (BDZ; FMO1 and FMO3 substrate) N- oxygenation was significantly decreased to 70% of individual controls. The expression levels of FMO1 and FMO3 proteins were not significantly changed in the sensitized mice. Hepatic inducible NO synthase (iNOS) mRNA level increased 5-fold and 15-fold in PS7 and SS7 mice, respectively, and hepatic tumor necrosis factor- α levels were greatly enhanced. When a selective iNOS inhibitor was injected into allergic mice, serum NO levels and BDZ N- oxygenation activity returned to control levels. NO directly suppressed BDZ N- oxygenation, which was probably related to FMO3-dependent metabolism in comparison with IMP N- oxidation. In hepatic microsomes from PS7 and SS7 mice, the suppression of BDZ N- oxygenation was restored by ascorbate. Therefore, type 1 allergic mice had differentially suppressed FMO3-dependent BDZ N- oxygenation. The suppression of FMO3 metabolism related to reversible S- nitrosyl modifications of iNOS-derived NO. NO is expected to alter FMO3-metabolic capacity-limited drug pharmacokinetics in humans. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

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Yang, Po-Min; Huang, Yu-Ting; Zhang, Yu-Qi; Hsieh, Chia-Wen; Wung, Being-Sun

2016-12-01

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25μM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca 2+ level. Furthermore, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca 2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca 2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser 1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25μM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser 1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca 2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization. Copyright © 2016 Elsevier Inc. All rights reserved.

Plasma oxidative stress level of IgA nephropathy in children and the effect of early intervention with angiotensin-converting enzyme inhibitors.

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Pei, Yuxin; Xu, Yuanyuan; Ruan, Jingwei; Rong, Liping; Jiang, Mengjie; Mo, Ying; Jiang, Xiaoyun

2016-01-01

The purpose of this study was to investigate the change of the plasma oxidative stress level in children with IgA nephropathy (IgAN) and analyze its relativity to the clinical and pathological classification. To discuss the early effects of angiotensin-converting enzyme inhibitors (ACEIs) on the plasma oxidative stress level in children with IgA nephropathy. Thirty-eight children with IgAN were divided into groups according to their clinical features, pathologic grades, and treatments. Twenty healthy children were included in the control group. The plasma level of advanced oxidation protein products (AOPPs), malonaldehyde (MDA), and superoxide dismutase (SOD) were detected. The plasma level of oxidative stress was significantly increased in the IgAN group, including a higher plasma level of AOPP and MDA and a lower plasma level of SOD. After treatment, the plasma level of oxidative stress was significantly decreased in the ACEI group. The children with IgAN had an increase in the plasma level of oxidative stress, expressed as an increased plasma level of AOPP and MDA and a decreased plasma level of SOD. Oxidative stress was associated with the progression of IgAN in children. Early treatment with ACEI therapy can significantly reduce the plasma level of oxidative stress in children with IgAN. © The Author(s) 2016.

Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: involvement of the adaptive antioxidant response.

Science.gov (United States)

Xue, Peng; Hou, Yongyong; Zhang, Qiang; Woods, Courtney G; Yarborough, Kathy; Liu, Huiyu; Sun, Guifan; Andersen, Melvin E; Pi, Jingbo

2011-04-08

There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 μM) inorganic arsenite (iAs³(+)) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs³(+) exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs³(+) exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs³(+) exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes. Copyright © 2011 Elsevier Inc. All rights reserved.

Mitochondrial oxidative stress causes hyperphosphorylation of tau.

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Simon Melov

2007-06-01

Full Text Available Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD: tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2 die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576 with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

Ob/ob mouse livers show decreased oxidative phosphorylation efficiencies and anaerobic capacities after cold ischemia.

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Michael J J Chu

Full Text Available BACKGROUND: Hepatic steatosis is a major risk factor for graft failure in liver transplantation. Hepatic steatosis shows a greater negative influence on graft function following prolonged cold ischaemia. As the impact of steatosis on hepatocyte metabolism during extended cold ischaemia is not well-described, we compared markers of metabolic capacity and mitochondrial function in steatotic and lean livers following clinically relevant durations of cold preservation. METHODS: Livers from 10-week old leptin-deficient obese (ob/ob, n = 9 and lean C57 mice (n = 9 were preserved in ice-cold University of Wisconsin solution. Liver mitochondrial function was then assessed using high resolution respirometry after 1.5, 3, 5, 8, 12, 16 and 24 hours of storage. Metabolic marker enzymes for anaerobiosis and mitochondrial mass were also measured in conjunction with non-bicarbonate tissue pH buffering capacity. RESULTS: Ob/ob and lean mice livers showed severe (>60% macrovesicular and mild (<30% microvesicular steatosis on Oil Red O staining, respectively. Ob/ob livers had lower baseline enzymatic complex I activity but similar adenosine triphosphate (ATP levels compared to lean livers. During cold storage, the respiratory control ratio and complex I-fueled phosphorylation deteriorated approximately twice as fast in ob/ob livers compared to lean livers. Ob/ob livers also demonstrated decreased ATP production capacities at all time-points analyzed compared to lean livers. Ob/ob liver baseline lactate dehydrogenase activities and intrinsic non-bicarbonate buffering capacities were depressed by 60% and 40%, respectively compared to lean livers. CONCLUSIONS: Steatotic livers have impaired baseline aerobic and anaerobic capacities compared to lean livers, and mitochondrial function indices decrease particularly from after 5 hours of cold preservation. These data provide a mechanistic basis for the clinical recommendation of shorter cold storage durations in

Exercise-induced oxidative stress and antioxidant enzyme activity in type 2 diabetic patients with and without diastolic dysfunction and hypertension

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Kostić Nada

2009-01-01

Full Text Available Introduction. Antioxidant systems are important factors affecting the oxidation of lipoproteins and thereby the progression of atherosclerotic disease. It has been suggested that physical activity might maintain and promote the antioxidant defence capacity against the oxidative stress. Left ventricular dysfunction (LVDD and hypertension are more common in subjects with diabetes mellitus (DM type 2. Objective. To evaluate the oxidative stress in patients with DM type 2, particularly with LVDD and hypertension and to determine the influence of acute exercise training on the investigated parameters. Methods. To assess the oxidative stress of patients, we determined the following antioxidative parameters: triglycerides (TG, total cholesterol, low density cholesterol, OxLDL cholesterol, superoxide dismutase (SOD, glutathione peroxidase (GSH-Px, plasminogen activator-type 1 (PAI-1 which were measured at rest and immediately after the acute bout of the cardiopulmonary exercise cycle ergometer test. Results. In basal conditions, diabetic patients had a significant increase of TG (3.12±1.09 vs 1.74±0.9 mmol/l; p<0.01, OxLDL cholesterol (84.73±16.9 vs 79.00±29.26 mmol/l; p<0.05 and SOD enzyme activity (913.38±120.36 vs 877.14 ±153.18; p<0.05 compared to controls. During the acute exercise test, there were significantly greater levels of OxLDL (84.73±16.90 vs 92.33±23.29 mmol/l; p<0.05 in study patients. SOD significantly increased in both groups during exercise, in diabetic patients (913.38±120.36 vs 921.50±130.03 U/g Hb; p<0.05 and in controls (877.14±153.18 vs 895.00±193.49 U/g Hb; p<0.05. GSH-Px significantly increased only in diabetic patients after acute exercise (45.04±11.19 vs 51.81±15.07 U/g Hb; p<0.01, but not in controls. PAI significantly decreased during the exercise test only in healthy subjects (2.60±0.35 vs 2.22±0.65; p<0.05. Type 2 diabetic patients with cardiovascular complications (LVDD and hypertension had a significant

Nutrigenetics and modulation of oxidative stress.

Science.gov (United States)

Da Costa, Laura A; Badawi, Alaa; El-Sohemy, Ahmed

2012-01-01

Oxidative stress develops as a result of an imbalance between the production and accumulation of reactive species and the body's ability to manage them using exogenous and endogenous antioxidants. Exogenous antioxidants obtained from the diet, including vitamin C, vitamin E, and carotenoids, have important roles in preventing and reducing oxidative stress. Individual genetic variation affecting proteins involved in the uptake, utilization and metabolism of these antioxidants may alter their serum levels, exposure to target cells and subsequent contribution to the extent of oxidative stress. Endogenous antioxidants include the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, paraoxanase, and glutathione S-transferase. These enzymes metabolize reactive species and their by-products, reducing oxidative stress. Variation in the genes coding these enzymes may impact their enzymatic antioxidant activity and, thus, the levels of reactive species, oxidative stress, and risk of disease development. Oxidative stress may contribute to the development of chronic disease, including osteoporosis, type 2 diabetes, neurodegenerative diseases, cardiovascular disease, and cancer. Indeed, polymorphisms in most of the genes that code for antioxidant enzymes have been associated with several types of cancer, although inconsistent findings between studies have been reported. These inconsistencies may, in part, be explained by interactions with the environment, such as modification by diet. In this review, we highlight some of the recent studies in the field of nutrigenetics, which have examined interactions between diet, genetic variation in antioxidant enzymes, and oxidative stress. Copyright © 2012 S. Karger AG, Basel.

Effect of temperature on oxidative stress parameters and enzyme activity in tissues of Cape river crab (Potamanautes perlatus) following exposure to silver nanoparticles (AgNPs)

CSIR Research Space (South Africa)

Walters, Chavon R

2016-01-01

Full Text Available of oxidative stress was studied in the gills and hepatopancreas of the Cape River crab Potamonautes perlatus. Responses were assessed through activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and the nonenzymatic...

Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks׳ gestation in nulliparous women who subsequently develop preeclampsia.

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Mistry, Hiten D; Gill, Carolyn A; Kurlak, Lesia O; Seed, Paul T; Hesketh, John E; Méplan, Catherine; Schomburg, Lutz; Chappell, Lucy C; Morgan, Linda; Poston, Lucilla

2015-01-01

Preeclampsia is a pregnancy-specific condition affecting 2-7% of women and a leading cause of perinatal and maternal morbidity and mortality. Deficiencies of specific micronutrient antioxidant activities associated with copper, selenium, zinc, and manganese have previously been linked to preeclampsia at the time of disease. Our aims were to investigate whether maternal plasma micronutrient concentrations and related antioxidant enzyme activities are altered before preeclampsia onset and to examine the dependence on genetic variations in these antioxidant enzymes. Predisease plasma samples (15±1 weeks׳ gestation) were obtained from women enrolled in the international Screening for Pregnancy Endpoints (SCOPE) study who subsequently developed preeclampsia (n=244) and from age- and BMI-matched normotensive controls (n=472). Micronutrient concentrations were measured by inductively coupled plasma mass spectrometry; associated antioxidant enzyme activities, selenoprotein-P, ceruloplasmin concentration and activity, antioxidant capacity, and markers of oxidative stress were measured by colorimetric assays. Sixty-four tag-single-nucleotide polymorphisms (SNPs) within genes encoding the antioxidant enzymes and selenoprotein-P were genotyped using allele-specific competitive PCR. Plasma copper and ceruloplasmin concentrations were modestly but significantly elevated in women who subsequently developed preeclampsia (both Ppreeclampsia. The modest elevation in copper may contribute to oxidative stress, later in pregnancy, in those women that go on to develop preeclampsia. The lack of evidence to support the hypothesis that functional SNPs influence antioxidant enzyme activity in pregnant women argues against a role for these genes in the etiology of preeclampsia. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

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Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Fibronectin phosphorylation by ecto-protein kinase

International Nuclear Information System (INIS)

Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

1988-01-01

The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

Protective Effects of Maillard Reaction Products of Whey Protein Concentrate against Oxidative Stress through an Nrf2-Dependent Pathway in HepG2 Cells.

Science.gov (United States)

Pyo, Min Cheol; Yang, Sung-Yong; Chun, Su-Hyun; Oh, Nam Su; Lee, Kwang-Won

2016-09-01

Whey protein concentrate (WPC), which contains α-lactalbumin and β-lactoglobulin, is utilized widely in the food industry. The Maillard reaction is a complex reaction that produces Maillard reaction products (MRPs), which are associated with the formation of antioxidant compounds. In this study, the hepatoprotection activity of MRPs of WPC against oxidative stress through the nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant pathway in HepG2 cells was examined. Glucose-whey protein concentrate conjugate (Glc-WPC) was obtained from Maillard reaction between WPC and glucose. The fluorescence intensity of Glc-WPC increased after 7 d compared to native WPC, and resulted in loss of 48% of the free amino groups of WPC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of Glc-WPC showed the presence of a high-molecular-weight portion. Treatment of HepG2 cells with Glc-WPC increased cell viability in the presence of oxidative stress, inhibited the generation of intracellular reactive oxygen species by tert-butyl hydroperoxide (t-BHP), and increased the glutathione level. Nrf2 translocation and Nrf2, reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-quinone oxidoreductase 1 (NOQ1), heme oxygenase-1 (HO-1), glutamate-L-cysteine ligase (GCL)M and GCLC mRNA levels were increased by Glc-WPC. Also, Glc-WPC increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). The results of this study demonstrate that Glc-WPC activates the Nrf2-dependent pathway through the phosphorylation of ERK1/2 and JNK in HepG2 cells, and induces production of antioxidant enzymes and phase II enzymes.

Exquisite Enzyme-Fenton Biomimetic Catalysts for Hydroxyl Radical Production by Mimicking an Enzyme Cascade.

Science.gov (United States)

Zhang, Qi; Chen, Shuo; Wang, Hua; Yu, Hongtao

2018-03-14

Hydrogen peroxide (H 2 O 2 ) is a key reactant in the Fenton process. As a byproduct of enzymatic reaction, H 2 O 2 can be obtained via catalytical oxidation of glucose using glucose oxidase in the presence of O 2 . Another oxidation product (gluconic acid) can suitably adjust the microenvironmental pH contributing to the Fe 3+ /Fe 2+ cycle in the Fenton reaction. Enzymes are extremely efficient at catalyzing a variety of reactions with high catalytic activity, substrate specificity, and yields in living organisms. Inspired by the multiple functions of natural multienzyme systems, an exquisite nanozyme-modified α-FeOOH/porous carbon (PC) biomimetic catalyst constructed by in situ growth of glucose oxidase-mimicking Au nanoparticles and crystallization of adsorbed ferric ions within carboxyl into hierarchically PC is developed as an efficient enzyme-Fenton catalyst. The products (H 2 O 2 , ∼4.07 mmol·L -1 ) of the first enzymatic reaction are immediately used as substrates for the second Fenton-like reaction to generate the valuable • OH (∼96.84 μmol·L -1 ), thus mimicking an enzyme cascade pathway. α-FeOOH nanocrystals, attached by C-O-Fe bondings, are encapsulated into the mesoporous PC frameworks, facilitating the electron transfer between α-FeOOH and the PC support and greatly suppressing iron leaching. This study paves a new avenue for designing biomimetic enzyme-based Fenton catalysts mimicking a natural system for • OH production.

Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

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Pallavi Sontakke

Full Text Available The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

Nitro-Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes.

Science.gov (United States)

Rosenblat, Mira; Rom, Oren; Volkova, Nina; Aviram, Michael

2016-08-01

Nitro-fatty acids possess anti-atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro-oleic acid (OLA-NO2) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti-oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA-NO2 (0-1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7-dichlorofluorescein diacetate, decreased dose-dependently, but the anti-oxidative effects of OLA-NO2 were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA-NO2 treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA-NO2 vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA-NO2 vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA-NO2 treated cells demonstrated down-regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone-sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA-NO2 vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein-mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA-NO2 modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti-oxidants and triglyceride

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y.

Science.gov (United States)

Vidau, Cyril; González-Polo, Rosa A; Niso-Santano, Mireia; Gómez-Sánchez, Rubén; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Blasco, Rafael; Brunet, Jean-Luc; Belzunces, Luc P; Fuentes, José M

2011-12-01

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

Enzyme/Mod. List: E0000000102 [jPOST repository metadata[Archive

Lifescience Database Archive (English)

Full Text Available E0000000102 Enzyme1 Trypsin Carbamidomethyl (C) Acetyl (N-term) Oxidation (M) Brucella abortus 104M The Enz...yme specificity was set to trypsin and a maximum of two missed cleavages were allow

Method for enzyme synthesis of radioactive thymine 5'-deoxyribonucleotides

International Nuclear Information System (INIS)

Nejedly, Z.; Ekl, J.; Hybs, K.; Kolina, J.; Filip, J.; Votruba, I.; Skoda, J.

1978-01-01

The enzyme synthesis is described for thymidine-5'-monophosphate, thymidine-5'-diphosphate and thymidine-5'-triphosphate specifically or nonspecifically labelled with 14 C or 3 H. The anabolic transformation of radioactive thymine to radioactive thymine 5'-deoxyribonucleotides is catalyzed by the action of enzyme preparations separated from Escherichia coli bacteria. It is achieved by the action of nonpurified cell-free extracts on special auxotrophic mutants of the thymine-dependent Escherichia coli SPT - strain in the presence of deoxyriboso-1-phosphate and adenosine-5'-triphosphate. The radioactive thymidine-5'-monophosphate may further be phosphorylated. In reaction mixtures, radioactive thymine, deoxyriboso-1-phosphate and adenosine-5'-triphosphate are used in molar ratios of 1:1:2 to 1:10:100, the optimum molar ratio being 1:5:10. (B.S.)

The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

Science.gov (United States)

Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

2016-01-01

The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

Nitric oxide: a physiologic messenger.

Science.gov (United States)

Lowenstein, C J; Dinerman, J L; Snyder, S H

1994-02-01

To review the physiologic role of nitric oxide, an unusual messenger molecule that mediates blood vessel relaxation, neurotransmission, and pathogen suppression. A MEDLINE search of articles published from 1987 to 1993 that addressed nitric oxide and the enzyme that synthesizes it, nitric oxide synthase. Animal and human studies were selected from 3044 articles to analyze the clinical importance of nitric oxide. Descriptions of the structure and function of nitric oxide synthase were selected to show how nitric oxide acts as a biological messenger molecule. Biochemical and physiologic studies were analyzed if the same results were found by three or more independent observers. Two major classes of nitric oxide synthase enzymes produce nitric oxide. The constitutive isoforms found in endothelial cells and neurons release small amounts of nitric oxide for brief periods to signal adjacent cells, whereas the inducible isoform found in macrophages releases large amounts of nitric oxide continuously to eliminate bacteria and parasites. By diffusing into adjacent cells and binding to enzymes that contain iron, nitric oxide plays many important physiologic roles. It regulates blood pressure, transmits signals between neurons, and suppresses pathogens. Excess amounts, however, can damage host cells, causing neurotoxicity during strokes and causing the hypotension associated with sepsis. Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

Magnetically responsive enzyme powders

Czech Academy of Sciences Publication Activity Database

Pospišková, K.; Šafařík, Ivo

2015-01-01

Roč. 380, APR 2015 (2015), s. 197-200 ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 2.357, year: 2015

Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

International Nuclear Information System (INIS)

Pendergast, A.M.

1986-01-01

The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

Effects of lemongrass oil and citral on hepatic drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in rats

Directory of Open Access Journals (Sweden)

Chien-Chun Li

2018-01-01

Full Text Available The essential oil from a lemongrass variety of Cymbopogon flexuosus [lemongrass oil (LO] is used in various food and aroma industry products and exhibits biological activities, such as anticancer and antimicrobial activities. To investigate the effects of 200 LO (200 mg/kg and 400 LO (400 mg/kg and its major component, citral (240 mg/kg, on drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in the liver, male Sprague-Dawley rats were fed a pelleted diet and administered LO or citral by gavage for 2 weeks. After 2 weeks of feeding, the effects of LO and citral on the metabolism and toxicity of acetaminophen were determined. The results showed that rats treated with 400 LO or citral had significantly reduced hepatic testosterone 6β-hydroxylation and ethoxyresorufin O-deethylation activities. In addition, NAD(PH:quinone oxidoreductase 1 activity was significantly increased by citral, and Uridine 5′-diphospho (UDP glucurosyltransferase activity was significantly increased by 400 LO in the rat liver. Treatment with 400 LO or citral reduced lipid peroxidation and reactive oxygen species levels in the liver. After acetaminophen treatment, however, LO and citral treatment resulted in little or no change in plasma alanine aminotransferase activity and acetaminophen-protein adducts content in the liver. Our results indicate that LO and citral may change the activities of drug-metabolizing enzymes and reduce oxidative stress in the liver. However, LO and citral may not affect the detoxification of acetaminophen.

Effect of Curcumin on Phosphorylation of AMPK and ACC in C2C12 Skeletal Muscle Cells

Directory of Open Access Journals (Sweden)

F Ghanbarzadeh

2013-10-01

Full Text Available Introduction: AMP activated protein kinase (AMPK as key regulators of cell metabolism, plays a major role in the activation of catabolic pathways, such as glucose transport and fatty acid oxidation. Thus, activation of this pathway can be used in the treatment of diabetes and metabolic syndrome. Many studied proposed the effectiveness of the polyphenols present in rhizomes of turmeric (curcumin on diabetes and its related complications. Therefore, this study investigated the effects of curcumin as an activator of AMPK pathway in C2C12 muscle cells. Methods: This study was done on C2C12 skeletal muscle cell line. The cells were classified into two distinct groups: first group was treated with 40µM curcumin and the second one with 0.1% DMSO as a negative control. The phosphorylated (AMPK and phosphorylated acetyl COA carboxylase (ACC were evaluated and compared by Western blotting technique. Results: intracellular phosphorylated AMPK protein content in Curcumin-treated group was 132.6% and ACC protein phosphorylated was 366.47%. Conclusion: This study showed that the levels of phosphorylated AMPK and ACC protein in cells treated with curcumin are higher than the negative control. Thus curcumin can be regarded as an activator of AMPK activity in these cells and can assist as a potential target for making anti diabetic medecine that has a synergistic activity with insulin.

Enzyme Histochemistry for Functional Histology in Invertebrates.

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Cima, Francesca

2017-01-01

In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.

Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger.

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Wang, Lu; Zhang, Jianhua; Cao, Zhanglei; Wang, Yajun; Gao, Qiang; Zhang, Jian; Wang, Depei

2015-01-16

The spore germination rate and growth characteristics were compared between the citric acid high-yield strain Aspergillus niger CGMCC 5751 and A. niger ATCC 1015 in media containing antimycin A or DNP. We inferred that differences in citric acid yield might be due to differences in energy metabolism between these strains. To explore the impact of energy metabolism on citric acid production, the changes in intracellular ATP, NADH and NADH/NAD+ were measured at various fermentation stages. In addition, the effects of antimycin A or DNP on energy metabolism and citric acid production was investigated by CGMCC 5751. By comparing the spore germination rate and the extent of growth on PDA plates containing antimycin A or DNP, CGMCC 5751 was shown to be more sensitive to antimycin A than ATCC 1015. The substrate-level phosphorylation of CGMCC 5751 was greater than that of ATCC 1015 on PDA plates with DNP. DNP at tested concentrations had no apparent effect on the growth of CGMCC 5751. There were no apparent effects on the mycelial morphology, the growth of mycelial pellets or the dry cell mass when 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP was added to medium at the 24-h time point. The concentrations of intracellular ATP, NADH and NADH/NAD+ of CGMCC 5751 were notably lower than those of ATCC 1015 at several fermentation stages. Moreover, at 96 h of fermentation, the citric acid production of CGMCC 5751 reached up to 151.67 g L(-1) and 135.78 g L(-1) by adding 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP, respectively, at the 24-h time point of fermentation. Thus, the citric acid production of CGMCC 5751 was increased by 19.89% and 7.32%, respectively. The concentrations of intracellular ATP, NADH and NADH/NAD+ of the citric acid high-yield strain CGMCC 5751 were notably lower than those of ATCC 1015. The excessive ATP has a strong inhibitory effect on citric acid accumulation by A. niger. Increasing NADH oxidation and appropriately reducing the concentration of

In vivo enzyme activity in inborn errors of metabolism

Energy Technology Data Exchange (ETDEWEB)

Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. (Clinical Research Centre, Harrow (England))

1990-08-01

Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

In vivo enzyme activity in inborn errors of metabolism

International Nuclear Information System (INIS)

Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D.

1990-01-01

Low-dose continuous infusions of [2H5]phenylalanine, [1-13C]propionate, and [1-13C]leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD

Caloric restriction mimetic 2-deoxyglucose maintains cytoarchitecture and reduces tau phosphorylation in primary culture of mouse hippocampal pyramidal neurons.

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Bele, M S; Gajare, K A; Deshmukh, A A

2015-06-01

Typical form of neurons is crucially important for their functions. This is maintained by microtubules and associated proteins like tau. Hyperphosphorylation of tau is a major concern in neurodegenerative diseases. Glycogen synthase kinase3β (GSK3β) and cyclin-dependent protein kinase 5 (Cdk5) are the enzymes that govern tau phosphorylation. Currently, efforts are being made to target GSK3β and Cdk5 as possible therapeutic avenues to control tau phosphorylation and treat neurodegenerative diseases related to taupathies. In a number of studies, caloric restriction mimetic 2-deoxyglucose (C6H12O5) was found to be beneficial in improving the brain functions. However, no reports are available on the effect of 2-deoxyglucose 2-DG on tau phosphorylation. In the present study, hippocampal pyramidal neurons from E17 mouse embryos were isolated and cultured on poly-L-lysine-coated coverslips. Neurons from the experimental group were treated with 10 mM 2-deoxyglucose. The treatment of 2-DG resulted in healthier neuronal morphology in terms of significantly lower number of cytoplasmic vacuoles, little or no membrane blebbings, maintained axon hillock and intact neurites. There were decreased immunofluorescence signals for GSK3β, pTau at Ser262, Cdk5 and pTau at Ser235 suggesting decreased tau phosphorylation, which was further confirmed by Western blotting. The results indicate the beneficial effects of 2-DG in controlling the tau phosphorylation and maintaining the healthy neuronal cytoarchitecture.

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

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Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

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José Maria Rodrigues da Luz

2012-12-01

Full Text Available The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse. The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase and hydrolytic enzymes (cellulases, xylanases and tanases. Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6. These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Phosphorylation of human skeletal muscle myosin

International Nuclear Information System (INIS)

Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

1986-01-01

Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

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Choi, Sung E; Kwon, Sanghoon; Seok, Sunmi; Xiao, Zhen; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Shinoda, Kosaku; Kajimura, Shingo; Kemper, Byron; Kemper, Jongsook Kim

2017-08-01

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases. Copyright © 2017 American Society for Microbiology.

The role of enzymes in fungus-growing ant evolution

DEFF Research Database (Denmark)

de Fine Licht, Henrik Hjarvard

behaviour. Here we report the first large-scale comparative study on fungus garden enzyme profiles and show that various interesting changes can be documented. A more detailed analysis of laccase expression, an enzyme that is believed to oxidize phenols in defensive secondary plant compounds such as tannins...

Endothelin-1 stimulates catalase activity through the PKCδ mediated phosphorylation of Serine 167

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