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Sample records for acid anhydrases

  1. Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish.

    Ruben Postel

    Full Text Available The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5 mutation, collapse of fins (cof, which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.

  2. Comparison of inhibition effects of some benzoic acid derivatives on sheep heart carbonic anhydrase

    Kiliç, Deryanur; Yildiz, Melike; Şentürk, Murat; Erdoǧan, Orhan; Küfrevioǧlu, Ömer Irfan

    2016-04-01

    Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3- and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. In the present study, inhibition of CA with some benzoic derivatives (1-6) were investigated. Sheep heart CA (shCA) enzyme was isolated by means of designed affinity chromatography gel (cellulose-benzyl-sulfanylamide) 42.45-fold in a yield of 44 % with 564.65 EU/mg. Purified shCA enzyme was used in vitro studies. In the studies, IC50 values were calculated for 3-aminobenzoic acid (1), 4-aminobenzoic acid (2), 2-hydroxybenzoic acid (3), 2-benzoylbenzoic acid (4), 2,3-dimethoxybenzoic acid (5), and 3,4,5-trimethoxybenzoic acid (6), showing the inhibition effects on the purified enzyme. Such molecules can be used as pioneer for discovery of novel effective CA inhibitors for medicinal chemistry applications.

  3. Oxygen-18 incorporation into malic acid during nocturnal carbon dioxide fixation in crassulacean acid metabolism plants: a new approach to estimating in vivo carbonic anhydrase activity

    Holtum, J.A.M.; Summons, R.; Roeske, C.A.; Comins, H.N.; O' Leary, M.H.

    1984-01-01

    Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with TC YO2, then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the ( TC) malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance or by mass spectrometry. Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum, 40 for Kalanchoee daigremontiana, and 100 or greater for Bryophyllum tubiflorum, Kalanchoee serrata, and Kalanchoae tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature. 37 references, 2 figures, 8 tables.

  4. Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor.

    Kalyanavenkataraman, Subhalakshmi; Nanjan, Pandurangan; Banerji, Asoke; Nair, Bipin G; Kumar, Geetha B

    2016-06-01

    Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9μM to as high as 333μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA. PMID:27038848

  5. T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

    Hallerdei, Janine; Scheibe, Renate J.; Parkkila, Seppo; Waheed, Abdul; Sly, William S.; Gros, Gerolf; Wetzel, Petra; Endeward, Volker

    2010-01-01

    We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and C...

  6. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

    Boone, Christopher D. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); Tu, Chingkuang [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)

    2014-06-01

    The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.

  7. Alkyl sulfonic acide hydrazides: Synthesis, characterization, computational studies and anticancer, antibacterial, anticarbonic anhydrase II (hCA II) activities

    O. Ozdemir, Ummuhan; İlbiz, Firdevs; Balaban Gunduzalp, Ayla; Ozbek, Neslihan; Karagoz Genç, Zuhal; Hamurcu, Fatma; Tekin, Suat

    2015-11-01

    Methane sulfonic acide hydrazide, CH3SO2NHNH2 (1), ethane sulfonic acide hydrazide, CH3CH2SO2NHNH2 (2), propane sulfonic acide hydrazide, CH3CH2CH2SO2NHNH2 (3) and butane sulfonic acide hydrazide, CH3CH2CH2CH2SO2NHNH2 (4) have been synthesized as homologous series and characterized by using elemental analysis, spectrophotometric methods (1H-13C NMR, FT-IR, LC-MS). In order to gain insight into the structure of the compounds, we have performed computational studies by using 6-311G(d, p) functional in which B3LYP functional were implemented. The geometry of the sulfonic acide hydrazides were optimized at the DFT method with Gaussian 09 program package. A conformational analysis of compounds were performed by using NMR theoretical calculations with DFT/B3LYP/6-311++G(2d, 2p) level of theory by applying the (GIAO) approach. The anticancer activities of these compounds on MCF-7 human breast cancer cell line investigated by comparing IC50 values. The antibacterial activities of synthesized compounds were studied against Gram positive bacteria; Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus faecalis ATCC 29212 and Gram negative bacteria; Escherichia coli ATCC 11230, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumonia ATCC 70063 by using the disc diffusion method. The inhibition activities of these compounds on carbonic anhydrase II enzyme (hCA II) have been investigated by comparing IC50 and Ki values. The biological activity screening shows that butane sulfonic acide hydrazide (4) has more activity than the others against tested breast cancer cell lines MCF-7, Gram negative/Gram positive bacteria and carbonic anhydrase II (hCA II) isoenzyme.

  8. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

    Janine Hallerdei

    Full Text Available We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  9. Heterocyclic compounds as carbonic anhydrase inhibitor.

    Husain, Asif; Madhesia, Diwakar

    2012-12-01

    The carbonic anhydrases (CAs, EC 4.2.1.1) constitute interesting targets for the design of pharmacological agents useful in the treatment or prevention of a variety of disorders such as, glaucoma, acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema, and obesity. A quite new and unexpected application of the CA inhibitors (CAIs) is with regard to their potential use in the management (imaging and treatment) of hypoxic tumors. A series of sulfonamides, including some clinically used derivatives like acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug, as well as the sulfamate antiepileptic drug topiramate have been reported to inhibit various human carbonic anhydrase isozyme. Various heterocyclic sulfonamides have been reported in this review with their potency to inhibit different carbonic anhydrases isozymes. PMID:21981003

  10. Salivary carbonic anhydrase isoenzyme VI

    Kivelä, Jyrki; Parkkila, Seppo; Parkkila, Anna-Kaisa; Leinonen, Jukka; Rajaniemi, Hannu

    1999-01-01

    The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body by catalysing the reversible reaction CO2+ H2O ⇌ HCO3−+ H+ (Davenport & Fisher, 1938; Davenport, 1939; Maren, 1967). Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian CA gene family. It is exclusively expressed in the serous acinar cells of the parotid and submandibular glands, from where it is secreted into the saliva. In this review, we will discuss recent advances in research focused on the physiological role of salivary CA VI in the oral cavity and upper alimentary canal. PMID:10523402

  11. Generation of nitric oxide from nitrite by carbonic anhydrase

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B;

    2009-01-01

    In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between...... bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced in...... effectively in catalysis. Taken together, our results reveal a novel nitrous anhydrase enzymatic activity of CA that would function to link the in vivo main end products of energy metabolism (CO2/H+) to the generation of vasoactive NO. The CA-mediated NO production may be important to the correlation between...

  12. Generation of nitric oxide from nitrite by carbonic anhydrase:

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank Bo;

    2009-01-01

    In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between...... bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced in...... effectively in catalysis. Taken together, our results reveal a novel nitrous anhydrase enzymatic activity of CA that would function to link the in vivo main end products of energy metabolism (CO2/H+) to the generation of vasoactive NO. The CA-mediated NO production may be important to the correlation between...

  13. Carbonic anhydrase IX in early-stage non-small cell lung cancer.

    Kim, S.; Rabbani, Z.N.; Vollmer, R.T.; Schreiber, E.G.; Oosterwijk, E.; Dewhirst, M.W.; Vujaskovic, Z.; Kelley, M.J.

    2004-01-01

    PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might al

  14. Increased oxidation-related glutathionylation and carbonic anhydrase activity in endometriosis.

    Andrisani, Alessandra; Donà, Gabriella; Brunati, Anna Maria; Clari, Giulio; Armanini, Decio; Ragazzi, Eugenio; Ambrosini, Guido; Bordin, Luciana

    2014-06-01

    permeability and adhesion molecule expression, thus contributing to ongoing inflammation. Due to their main cellular functions--delivery of O2 from lung to tissue and removal of CO2 from tissue to lung--red blood cells (RBC) are exposed to oxidative stress. Carbon dioxide in tissue capillaries diffuses into red cells, where it is rapidly hydrated by the action of cytosolic carbonic anhydrase. Analysis of the oxidation status of endometriotic RBC membranes showed a high content of glutathionylated proteins, indicating pre-existing oxidation-related alterations. The increase in glutathionylated proteins was correlated to increased carbonic anhydrase activity in endometriotic RBC compared with healthy controls. Carbonic anhydrase is a family of metalloenzymes involved in many physiological processes such as acid-base homeostasis, respiration, carbon dioxide and ion transport, and bone resorption, and in the regulation of ureagenesis, gluconeogenesis, lipogenesis and tumourigenesis. Due to the potential implication of carbonic anhydrase activation in many pathologies, such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis to prevent possible complications and/or worsening of related conditions. PMID:24746440

  15. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    In a new assay of carbonic anhydrase, NaH14CO3 solution at the bottom of a sealed vessel releases 14CO3 which diffuses to the top of the vessel to be assimilated by actively photosynthesizing Chlamydomonas cells. The assay is initiated by illuminating cells and stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid stable radioactivity above the uncatalyzed background level. With bovine carbonic anhydrase, 1.5 Wilbur Anderson Unit (WAU) can be consistantly measured at 5-6 fold above background. Sonicated whole cells of air adapted wild type (+)gave 741.1 ± 12.4 WAU/mg chl. Intact washed cells of mixotrophically grown wall-less mutant CWD(-) and a high CO2 requiring wall-less double mutant CIA-3/CW15 (-) gave 7.1 ± 1.9 and 2.8 ± 7.8 WAU/mg chl respectively. Chloroplasts isolated from CWD and CIA-3/CW15 and subsequently disrupted gave 64.0 ± 14.7 and 2.8 ± 3.2 WAU/mg chl respectively. Chloroplast sonicate from another wall-less mutant CW15(-) gave activity comparable to CWD. Thus on a chlorophyll basis, enzyme activity in chloroplasts from mixotrophically grown cells is about 1/10th of the level found in air adapted wild type cells. CIA-3 seems to lack this activity

  16. Carbonic anhydrase inhibitors drug design.

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported. PMID:24146385

  17. Atomic resolution studies of carbonic anhydrase II

    The structure of human carbonic anhydrase II has been solved with a sulfonamide inhibitor at 0.9 Å resolution. Structural variation and flexibility is seen on the surface of the protein and is consistent with the anisotropic ADPs obtained from refinement. Comparison with 13 other atomic resolution carbonic anhydrase structures shows that surface variation exists even in these highly ordered isomorphous crystals. Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 Å resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 Å) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 Å in some zinc–protein and zinc–ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available

  18. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  19. How many carbonic anhydrase inhibition mechanisms exist?

    Supuran, Claudiu T

    2016-01-01

    Six genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) were described to date. Inhibition of CAs has pharmacologic applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents. New classes of CA inhibitors (CAIs) were described in the last decade with enzyme inhibition mechanisms differing considerably from the classical inhibitors of the sulfonamide or anion type. Five different CA inhibition mechanisms are known: (i) the zinc binders coordinate to the catalytically crucial Zn(II) ion from the enzyme active site, with the metal in tetrahedral or trigonal bipyramidal geometries. Sulfonamides and their isosters, most anions, dithiocarbamates and their isosters, carboxylates, and hydroxamates bind in this way; (ii) inhibitors that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, 2-thioxocoumarins, sulfocoumarins); (iii) inhibitors which occlude the entrance to the active site cavity (coumarins and their isosters), this binding site coinciding with that where CA activators bind; (iv) compounds which bind out of the active site cavity (a carboxylic acid derivative was seen to inhibit CA in this manner), and (v) compounds for which the inhibition mechanism is not known, among which the secondary/tertiary sulfonamides as well as imatinib/nilotinib are the most investigated examples. As CAIs are used clinically in many pathologies, with a sulfonamide inhibitor (SLC-0111) in Phase I clinical trials for the management of metastatic solid tumors, this review updates the recent findings in the field which may be useful for a structure-based drug design approach of more selective/potent modulators of the activity of these enzymes. PMID:26619898

  20. Bortezomib inhibits bacterial and fungal β-carbonic anhydrases.

    Supuran, Claudiu T

    2016-09-15

    Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from pathogenic fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, Malassezia globosa) and bacteria (three isoforms from Mycobacterium tuberculosis, Rv3273, Rv1284 and Rv3588), as well from the insect Drosophila melanogaster (DmeCA) and the plant Flaveria bidentis (FbiCA1) with the boronic acid peptidomimetic proteosome inhibitor bortezomib was investigated. Bortezomib was a micromolar inhibitor of all these enzymes, with KIs ranging between 1.12 and 11.30μM. Based on recent crystallographic data it is hypothesized that the B(OH)2 moiety of the inhibitor is directly coordinated to the zinc ion from the enzyme active site. The class of boronic acids, an under-investigated type of CA inhibitors, may lead to the development of anti-infectives with a novel mechanism of action, based on the pathogenic organisms CA inhibition. PMID:27469982

  1. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

    Didier Zoccola

    2016-06-01

    Full Text Available Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1 a change in gene expression under OA (2 an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

  2. Carborane-based carbonic anhydrase inhibitors

    Brynda, Jiří; Mader, Pavel; Šícha, Václav; Fábry, Milan; Poncová, Kristýna; Bakardjiev, Mario; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2013-01-01

    Roč. 52, č. 51 (2013), s. 13760-13763. ISSN 1433-7851 R&D Projects: GA TA ČR(CZ) TE01020028; GA AV ČR IAAX00320901 Institutional support: RVO:68378050 ; RVO:61388963 ; RVO:61388980 Keywords : carbonic anhydrases * carboranes * drug discovery * inhibitors * structure elucidation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 11.336, year: 2013

  3. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T; Tambutté, Sylvie

    2016-01-01

    Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity. PMID:27271641

  4. Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations.

    Carta, Fabrizio; Aggarwal, Mayank; Maresca, Alfonso; Scozzafava, Andrea; McKenna, Robert; Supuran, Claudiu T.

    2012-01-01

    The zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) is inhibited by several classes of zinc-binders (sulfonamides, sulfamates, and sulfamides) as well as by compounds which do not interact with the metal ion (phenols, polyamines and coumarins). Here we report a new class of potent CA inhibitors which bind the zinc ion: the dithiocarbamates (DTCs). They coordinate to the zinc ion from the enzyme active site in monodentate manner and establish many favorable interactions with amino acid residue...

  5. Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours

    Antti J. Kivel(a); Jyrki Kivel(a); Juha Saarnio; Seppo Parkkila

    2005-01-01

    Carbonic anhydrases (CAs) catalyse the hydration of CO2to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. This review is focused on the distribution and role of CA isoenzymes in both normal and pathological gastrointestinal (GI) tract tissues. It has been known for many years that CAs are widely present in the GI tract and play important roles in several physiological functions such as production of saliva, gastric acid, bile, and pancreatic juice as well as in absorption of salt and water in intestine. New information suggests that these enzymes participate in several processes that were not envisioned earlier. Especially, the recent reports on plasma membranebound isoenzymes Ⅸ and Ⅻ have raised considerable interest since they were reported to participate in cancer invasion and spread. They are induced by tumour hypoxia and may also play a role in von Hippel-Lindau (VHL)-mediated carcinogenesis.

  6. Hyperkalaemia induced by carbonic anhydrase inhibitor.

    Wakabayashi, Y.

    1991-01-01

    An 81-year-old man developed hyperkalaemic and hyperchloraemic metabolic acidosis following treatment with a carbonic anhydrase inhibitor for his glaucoma. He had mild renal failure and selective aldosterone deficiency was confirmed. In this case the treatment did not lead to hypokalaemia because of the limited potassium secretory capacity in the renal tubules from selective aldosterone deficiency; rather, it may have led to hyperkalaemia because metabolic acidosis induced by the carbonic anh...

  7. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Anna Di Fiore; Vincenzo Alterio; Simona M. Monti; Giuseppina De Simone; Katia D'Ambrosio

    2015-01-01

    Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques,...

  8. Carborane-based inhibitors of carbonic anhydrases

    Brynda, Jiří; Pachl, Petr; Šícha, Václav; Fábry, Milan; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2015-01-01

    Roč. 22, č. 1 (2015), s. 3. ISSN 1211-5894. [Discussions in Structural Molecular Biology . Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] R&D Projects: GA ČR GA15-05677S Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:61388980 Keywords : carboranes * carbonic anhydrase Subject RIV: CE - Biochemistry

  9. Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells

    Hu, Honghong

    2009-12-13

    The continuing rise in atmospheric CO2 causes stomatal pores in leaves to close and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress. However, the CO2-binding proteins that control this response remain unknown. Moreover, which cell type responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate. We demonstrate that Arabidopsis thaliana double-mutant plants in the beta-carbonic anhydrases betaCA1 and betaCA4 show impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses. betaCA-mediated CO2-triggered stomatal movements are not, in first-order, linked to whole leaf photosynthesis and can function in guard cells. Furthermore, guard cell betaca-overexpressing plants exhibit instantaneous enhanced water use efficiency. Guard cell expression of mammalian alphaCAII complements the reduced sensitivity of ca1 ca4 plants, showing that carbonic anhydrase-mediated catalysis is an important mechanism for betaCA-mediated CO2-induced stomatal closure and patch clamp analyses indicate that CO2/HCO3- transfers the signal to anion channel regulation. These findings, together with ht1-2 (ref. 9) epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway, which controls gas-exchange between plants and the atmosphere.

  10. Accelerating Mineral Carbonation Using Carbonic Anhydrase.

    Power, Ian M; Harrison, Anna L; Dipple, Gregory M

    2016-03-01

    Carbonic anhydrase (CA) enzymes have gained considerable attention for their potential use in carbon dioxide (CO2) capture technologies because they are able to catalyze rapidly the interconversion of aqueous CO2 and bicarbonate. However, there are challenges for widespread implementation including the need to develop mineralization process routes for permanent carbon storage. Mineral carbonation of highly reactive feedstocks may be limited by the supply rate of CO2. This rate limitation can be directly addressed by incorporating enzyme-catalyzed CO2 hydration. This study examined the effects of bovine carbonic anhydrase (BCA) and CO2-rich gas streams on the carbonation rate of brucite [Mg(OH)2], a highly reactive mineral. Alkaline brucite slurries were amended with BCA and supplied with 10% CO2 gas while aqueous chemistry and solids were monitored throughout the experiments (hours to days). In comparison to controls, brucite carbonation using BCA was accelerated by up to 240%. Nesquehonite [MgCO3·3H2O] precipitation limited the accumulation of hydrated CO2 species, apparently preventing BCA from catalyzing the dehydration reaction. Geochemical models reproduce observed reaction progress in all experiments, revealing a linear correlation between CO2 uptake and carbonation rate. Data demonstrates that carbonation in BCA-amended reactors remained limited by CO2 supply, implying further acceleration is possible. PMID:26829491

  11. Metabolic Effect of Estrogen Receptor Agonists on Breast Cancer Cells in the Presence or Absence of Carbonic Anhydrase Inhibitors

    Anissa Belkaid

    2016-05-01

    Full Text Available Metabolic shift is one of the major hallmarks of cancer development. Estrogen receptor (ER activity has a profound effect on breast cancer cell growth through a number of metabolic changes driven by its effect on transcription of several enzymes, including carbonic anhydrases, Stearoyl-CoA desaturase-1, and oncogenes including HER2. Thus, estrogen receptor activators can be expected to lead to the modulation of cell metabolism in estrogen receptor positive cells. In this work we have investigated the effect of 17β-estradiol, an ER activator, and ferulic acid, a carbonic anhydrase inhibitor, as well as ER activator, in the absence and in the presence of the carbonic anhydrase inhibitor acetazolamide on the metabolism of MCF7 cells and MCF7 cells, stably transfected to express HER2 (MCF7HER2. Metabolic profiles were studied using 1D and 2D metabolomic Nuclear Magnetic Resonance (NMR experiments, combined with the identification and quantification of metabolites, and the annotation of the results in the context of biochemical pathways. Overall changes in hydrophilic metabolites were largest following treatment of MCF7 and MC7HER2 cells with 17β-estradiol. However, the carbonic anhydrase inhibitor acetazolamide had the largest effect on the profile of lipophilic metabolites.

  12. Future Perspective in Carbonic Anhydrase Inhibitors and its Drugs

    S.Petchimuthu; Dr. N. Narayanan

    2013-01-01

    Through this review it is contemplated that carbonic anhydrase inhibitors, were a traditional drugs of choice for the treatment of glaucoma with a myriad of side effects and inadequate topical effectiveness, may be formulated into a topically effective agent by utilizing various newer formulation approaches of ocular drug delivery. Even though the carbonic anhydrase inhibitor, acetazolamide (ACZ) has a poor solubility and penetration power (BCS Class IV), various studies mentioned in the revi...

  13. Carbonic Anhydrases and Their Biotechnological Applications

    Robert McKenna

    2013-08-01

    Full Text Available The carbonic anhydrases (CAs are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II has a catalytic efficiency of 108 M−1 s−1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development.

  14. Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B.

    Kandel, M; Gornall, A G; Lam, L K; Kandel, S I

    1975-05-01

    Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis. PMID:237619

  15. The effects of some avermectins on bovine carbonic anhydrase enzyme.

    Kose, Leyla Polat; Gülçin, İlhami; Özdemir, Hasan; Atasever, Ali; Alwasel, Saleh H; Supuran, Claudiu T

    2016-10-01

    Avermectins are effective agricultural pesticides and antiparasitic agents that are widely employed in the agricultural, veterinary and medical fields. The aim of this study was to investigate the inhibitory effects of selected avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin that are used as drugs against a wide variety of internal and external mammalian parasites, on the carbonic anhydrase enzyme (CA, EC 4.2.1.1.) purified from fresh bovine erythrocyte. CA catalyses the rapid interconversion of carbon dioxide (CO2) and water (H2O) to bicarbonate ([Formula: see text]) and protons (H(+)) and regulate the acidity of the local tissues. Bovine erythrocyte CA (bCA) enzyme was purified by Sepharose-4B affinity chromatography with a yield of 21.96% and 262.7-fold purification. The inhibition results obtained from this study showed Ki values of 9.73, 17.39, 20.43, 13.39, 16.44 and 17.73 nM for abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin, respectively. However, acetazolamide, well-known clinically established CA inhibitor, possessed a Ki value of 27.68 nM. PMID:26207514

  16. Thermodynamics of binding of Zn2+ to carbonic anhydrase inhibitors

    Remko, Milan; Garaj, Vladimír

    The Becke3LYP functional of DFT theory and the two-layered ONIOM (B3LYP/6-311+G(d,p): MNDO) method were used to characterize 46 gas-phase complexes of 34 neutral and anionic ligands (H2O, CH3OH, CH3COOH, CH3CONH2, HOSO2NH2, CO2, HSO2NH2, CH3SO2NH2, CH3C(=O)NHOH, imidazole, NH2SO2NH2, anions of 4-aminobenzenesulphonamide, saccharin, 1I9L, brinzolamide, dorzolamide, acetazolamide, further HO(-), CH3O(-), CH3COO(-), CH3CONH(-), N=N=N(-), S=C=N(-), CH3C(=O)NHO(-), HOCOO(-), imidazoleN(-), phenol-O(-), HOSO2NH(-), (-)OSO2NH(-), (-)OSO2NH2, H2NSO2NH(-), HSO2NH(-), CH3SO2NH(-), and CF3SO2NH(-), respectively) with Zn2+. Proton dissociation enthalpies and Gibbs energies of acidic inhibitors in the presence of zinc were computed. Their gas-phase acidity considerably increases upon chelation. Of the bases investigated, the weakest zinc affinity is exhibited by carbon dioxide (-313.5 kJ mol-1). Deprotonated inhibitors have higher affinities for zinc than the neutral ones. Compared to the other mono-deprotonated ligands the acetohydroxamic acid anion has the highest affinity for zinc (-1872.7 kJ mol-1). The zinc affinity of the acetazolamide anion computed using the hybrid ONIOM (B3LYP/6-311+G(d,p): MNDO) method is in very good agreement with the full DFT ones and this method can be adopted to model large complexes of inhibitors with the active site of carbonic anhydrase.

  17. Carbonic anhydrase inhibitors: Synthesis, characterization and inhibition activities of furan sulfonylhydrazones against carbonic anhydrase I (hCA I)

    Gündüzalp, Ayla Balaban; Parlakgümüş, Gökhan; Uzun, Demet; Özmen, Ümmuhan Özdemir; Özbek, Neslihan; Sarı, Musa; Tunç, Tuncay

    2016-02-01

    The methane sulfonic acide hydrazide (1) was used to obtain furan sulfonylhydrazones; 2-acetylfuranmethanesulfonylhydrazone (2), 2-furaldehydemethanesulfonylhydrazone (3), 5-nitro-2-furaldehydemethanesulfonylhydrazone (4). The structures of furan sulfonylhydrazones were determined by using elemental analysis, FT-IR, 1H NMR, 13C NMR and UV-vis methods. The structure of 5-nitro-2-furaldehydemethanesulfonylhydrazone (4) was also supported with X-ray difraction method and found that compound 4 was crystallized in triclinic, space group P 1 bar . In order to gain insight into the structure of the compounds, we performed computational studies by using 6-311G(d,p) basic set in which B3LYP correlation function was implemented. The geometry of the sulfonylhydrazones were optimized at DFT method with Gaussian 09 program package and the global reactivity descriptors were also calculated by this basic set. The enzyme inhibition activities of the sulfonylhydrazones were investigated on carbonic anhydrase I (hCA I) isoenzyme and their activity parameters (Km, IC50 and Ki) were calculated by spectrophotometric method. And also, their inhibitor effects were also investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Inhibition results show that compound 4 containing electron withdrawing group (NO2) has higher inhibition effect on hCA I isoenzyme than other's.

  18. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

    2016-01-01

    Abstract Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4. PMID:27560991

  19. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets.

    Supuran, Claudiu T

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO₂ hydration, with kcat values in the range of (3.4-8.3) × 10⁵ s(-1) and kcat/KM values of (4.7-8.5) × 10⁷ M(-1)·s(-1). In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2-88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets. PMID:27322334

  20. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    Claudiu T. Supuran

    2016-06-01

    Full Text Available Carbonic anhydrases (CAs, EC 4.2.1.1 are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3 × 105 s−1 and kcat/KM values of (4.7–8.5 × 107 M−1·s−1. In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM. The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2–88.5 nM. Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets.

  1. Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate.

    Wang, Xiaohong; Schröder, Heinz C; Schlossmacher, Ute; Neufurth, Meik; Feng, Qingling; Diehl-Seifert, Bärbel; Müller, Werner E G

    2014-05-01

    Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO₃ is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO₃ formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO₃ (-)) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca²⁺ salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO₃ deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive-negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects. PMID:24374859

  2. Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II

    This article reports the production, crystallization and X-ray structure determination of perdeuterated human carbonic anhydrase (HCA II). The refined structure is shown to be highly isomorphous with hydrogenated HCA II, especially with regard to the active site architecture and solvent network. Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH−) and the proton-shuttling residue His64. This distance (∼7.5 Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (∼1.0 Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography

  3. Structure and function of carbonic anhydrases.

    Supuran, Claudiu T

    2016-07-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyse the interconversion between CO2 and bicarbonate as well as other hydrolytic reactions. Among the six genetic families known to date, the α-, β-, γ-, δ-, ζ- and η-CAs, detailed kinetic and X-ray crystallographic studies have allowed a deep understanding of the structure-function relationship in this superfamily of proteins. A metal hydroxide nucleophilic species of the enzyme, and a unique active site architecture, with half of it hydrophilic and the opposing part hydrophobic, allow these enzymes to act as some of the most effective catalysts known in Nature. The CA activation and inhibition mechanisms are also known in detail, with a large number of new inhibitor classes being described in the last years. Apart from the zinc binders, some classes of inhibitors anchor to the metal ion coordinated nucleophile, others occlude the entrance of the active site cavity and more recently, compounds binding outside the active site were described. CA inhibition has therapeutic applications for drugs acting as diuretics, antiepileptics, antiglaucoma, antiobesity and antitumour agents. Targeting such enzymes from pathogens may lead to novel anti-infectives. Successful structure-based drug design campaigns allowed the discovery of highly isoform selective CA inhibitors (CAIs), which may lead to a new generation of drugs targeting these widespread enzymes. The use of CAs in CO2 capture processes for mitigating the global temperature rise has also been investigated more recently. PMID:27407171

  4. Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-{gamma}2

    Mitterberger, Maria C. [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria); Kim, Geumsoo [Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012 (United States); Rostek, Ursula [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria); Levine, Rodney L. [Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012 (United States); Zwerschke, Werner, E-mail: werner.zwerschke@oeaw.ac.at [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria)

    2012-05-01

    Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO{sub 2} have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII{sup -/-} MEFs compared with CAIII{sup +/+} cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-{gamma}2 (PPAR{gamma}2) and CCAAT/enhancer binding protein-{alpha}. We found a considerable (approximately 1000-fold) increase in the PPAR{gamma}2 expression in the CAIII{sup -/-} MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPAR{gamma}2 and FABP4. When both CAIII and PPAR{gamma}2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPAR{gamma}2 gene expression. -- Highlights: Black-Right-Pointing-Pointer We discover a novel function of Carbonic anhydrase III (CAIII). Black-Right-Pointing-Pointer We show that CAIII is a regulator of adipogenesis. Black-Right-Pointing-Pointer We demonstrate that CAIII acts at the level of PPAR{gamma}2 gene expression. Black-Right-Pointing-Pointer Our data contribute to a better understanding of the role of CAIII in fat tissue.

  5. Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2

    Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO2 have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII−/− MEFs compared with CAIII+/+ cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII−/− MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression. -- Highlights: ► We discover a novel function of Carbonic anhydrase III (CAIII). ► We show that CAIII is a regulator of adipogenesis. ► We demonstrate that CAIII acts at the level of PPARγ2 gene expression. ► Our data contribute to a better understanding of the role of CAIII in fat tissue.

  6. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  7. Androgen-linked control of rat liver carbonic anhydrase III.

    Shiels, A.; Jeffery, S; Phillips, I. R.; Shephard, E A; Wilson, C. A.; Carter, N D

    1983-01-01

    The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.

  8. Novel carborane based inhibitors of carbonic anhydrase IX

    Štěpánková, J.; Řezáčová, Pavlína; Brynda, Jiří; Harvanová, M.; Mašek, V.; Nová, A.; Siller, M.; Das, V.; Doležal, D.; Grüner, Bohumír; Šícha, Václav; Konečný, P.; Znojek, P.; Džubák, P.; Hajdúch, M.

    2015-01-01

    Roč. 75, 15 Suppl (2015), s. 4492. ISSN 0008-5472. [Annual Meeting of the American Association for Cancer Research (AACR) /106./. 18.04.2015-22.04.2015, Philadelphia] Institutional support: RVO:61388963 ; RVO:61388980 Keywords : carbonic anhydrase * carborane based inhibitors Subject RIV: CE - Biochemistry

  9. Carbonic anhydrases as targets for medicinal chemistry.

    Supuran, Claudiu T; Scozzafava, Andrea

    2007-07-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc enzymes acting as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate. 16 different alpha-CA isoforms were isolated in mammals, where they play crucial physiological roles. Some of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is secreted in saliva and milk. Three acatalytic forms are also known, the CA related proteins (CARP), CARP VIII, CARP X and CARP XI. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited primarily by two classes of compounds: the metal complexing anions and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO(2)NH(2) (R=aryl; hetaryl; perhaloalkyl; X=nothing, O or NH). Several important physiological and physio-pathological functions are played by CAs present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms represents an attractive goal for the design of inhibitors with biomedical applications. Indeed, CA inhibitors are clinically used as

  10. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, Sep 18 (2015), s. 223-232. ISSN 0223-5234 Institutional support: RVO:61388963 Keywords : human carbonic anhydrase * isoquinoline * quinoline * X-ray * molecular docking Subject RIV: CE - Biochemistry Impact factor: 3.447, year: 2014

  11. Membrane carbonic anhydrase (IV) and ciliary epithelium. Carbonic anhydrase activity is present in the basolateral membranes of the non-pigmented ciliary epithelium of rabbit eyes.

    Matsui, H; Murakami, M; Wynns, G C; Conroy, C W; Mead, A; Maren, T H; Sears, M L

    1996-04-01

    Carbonic anhydrase inhibitors (CAIs) lower intraocular pressure by reducing aqueous flow. It has been thought that this pharmacologic reduction of aqueous flow is mediated by the ciliary epithelium, but it is not known whether this cellular action is effected by inhibition of the membranal (CA IV) and/or cytosolic (CA II) carbonic anhydrases of the ciliary epithelium. The isolated ciliary epithelial bilayer maintains its anatomic and functional polarity and generates a transepithelial potential difference (TEP) in an Ussing type chamber. Depletion of HCO3-, accomplished either with an HCO3(-)-free solution bathing the epithelial bilayer, or, with addition of freely permeant CAIs to HCO3(-)-containing media, (from either the PE or NPE side of the bilayer) depolarizes the preparation. Addition of CAIs to an HCO3(-)-depleted preparation has no further effect, indicating the specific action of the CAIs. The CAI, 2-p-NH2 benzenesulfonamido-1,3,4,-thiadiazole-5-SO2NH2, linked to polybutadiene maleic acid yields an impermeant polymer of 20000 Da with no loss of activity. At 45 microM this impermeant polymer caused a 60% increase in the SCC, seen only when the compound was applied to the NPE side of the bilayer. This latter result indicates an effect from inhibition of CA IV in the basolateral membranes of the NPE. Thus there are probably two different cellular actions of CAIs upon the ciliary epithelium to reduce aqueous inflow, cytoplasmic and membranal. The action of NPE basolateral membranal CA IV is probably linked to the chloride/bicarbonate exchanger. PMID:8795459

  12. Revisiting Zinc Coordination in Human Carbonic Anhydrase II

    Song, He; Wilson, David L.; Farquhar, Erik R.; Lewis, Edwin A.; Emerson, Joseph P.

    2012-01-01

    Carbonic anhydrase (CA) is a well-studied, zinc-dependent metalloenzyme that catalyzes the hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA) is relatively easy to generate, and the reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka ~ 1.2×1012) for Zn2+. However, new reactivity data and isotherma...

  13. Structural analysis of inhibitor binding to human carbonic anhydrase II.

    Boriack-Sjodin, P. A.; Zeitlin, S; Chen, H H; Crenshaw, L.; Gross, S.; Dantanarayana, A.; P. Delgado; May, J. A.; Dean, T.; Christianson, D. W.

    1998-01-01

    X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bi...

  14. Variable involvement of the perivascular retinal tissue in carbonic anhydrase inhibitor induced relaxation of porcine retinal arterioles in vitro

    Kehler, Anne Katrine; Holmgaard, Kim; Hessellund, Anders;

    2007-01-01

    in a myograph. After precontraction with the prostaglandin analogue U46619, the vasorelaxing effect of the carbonic anhydrase inhibitors methyl bromopyruvate, ethyl bromopyruvate, acetazolamide, and dorzolamide were studied. RESULTS: All the examined carbonic anhydrase inhibitors induced a...

  15. Future Perspective in Carbonic Anhydrase Inhibitors and its Drugs

    S.Petchimuthu

    2013-09-01

    Full Text Available Through this review it is contemplated that carbonic anhydrase inhibitors, were a traditional drugs of choice for the treatment of glaucoma with a myriad of side effects and inadequate topical effectiveness, may be formulated into a topically effective agent by utilizing various newer formulation approaches of ocular drug delivery. Even though the carbonic anhydrase inhibitor, acetazolamide (ACZ has a poor solubility and penetration power (BCS Class IV, various studies mentioned in the review indicate that it is possible to successfully formulate topically effective ACZ by using:(i High concentration of the drug, (ii Surfactant gel preparations of ACZ, (iii ACZ loaded into liposomes, (iv Cyclodextrins to increase the solubility and hence bioavailability of ACZ, and Viscolyzers and other polymers either alone or in combination with cyclodextrins. With the advent of newer topical carbonic anhydrase inhibitors (CAIs like dorzolamide and brinzolamide, a localized effect with fewer side effects is expected.But whenever absorbed systemically, a similar range of adverse effects (attributable to sulphonamides may occur upon use. Furthermore, oral ACZ is reported to be more physiologically effective than 2% dorzolamide hydrochloridead ministered topically, even though in isolated tissues dorzolamide appears to be the most active as it shows the lowest IC50 values for CA-II and CA-IV. Hence, there exists considerable scope for the development of more/equally effective and inexpensive topically effective formulations of ACZ. The use of various formulation technologies discussed in this review can provide a fresh impetus to research in this area.

  16. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, SEP 18 (2015), s. 223-232. ISSN 0223-5234 R&D Projects: GA ČR GA15-05677S Grant ostatní: Fondo di Ateneo per la Ricerca (PRA)(IT) ORME09SPNC Institutional support: RVO:68378050 Keywords : Human carbonic anhydrase * Isoquinoline * Quinoline * X-ray * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.447, year: 2014

  17. Carbonic anhydrase II deficiency: Single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients

    Hu, P.Y.; Ernst, A.R.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (United States)); Venta, P.J. (Michigan State Univ., East Lansing, MI (United States)); Skaggs, L.A.; Tashian, R.E. (Univ. of Michigan Medical School, Ann Arbor, MI (United States))

    1994-04-01

    To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. The authors report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation. These findings identify a novel mutation common to Hispanic patients from the Caribbean islands and provide a ready means for PCR-based diagnosis of the [open quotes]Hispanic mutation.[close quotes] The basis for their phenotypic variability is not yet clear. 15 refs., 5 figs., 1 tab.

  18. Carbonic anhydrase activators: gold nanoparticles coated with derivatized histamine, histidine, and carnosine show enhanced activatory effects on several mammalian isoforms.

    Saada, Mohamed-Chiheb; Montero, Jean-Louis; Vullo, Daniela; Scozzafava, Andrea; Winum, Jean-Yves; Supuran, Claudiu T

    2011-03-10

    Lipoic acid moieties were attached to amine or amino acids showing activating properties against the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of histamine, L-histidine methyl ester, and L-carnosine methyl ester were attached to gold nanoparticles (NPs) by reaction with Au(III) salts in reducing conditions. The CA activators (CAAs)-coated NPs showed low nanomolar activation (K(A)s of 1-9 nM) of relevant cytosolic, membrane-bound, mitochondrial, and transmembrane CA isoforms, such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively activated CAs ex vivo, in whole blood experiments, with an increase of 200-280% of the CA activity. This is the first example of enzyme activation with nanoparticles and may lead to biomedical applications for conditions in which the CA activity is diminished, such as aging, Alzheimer's disease, or CA deficiency syndrome. PMID:21291238

  19. Sarcoidosis patient: an unexpected reaction to carbonic anhydrase enzyme inhibitor

    Khedr, Yahya A H; Khedr, Abdulla H

    2013-01-01

    Ocular diseases are very common in many of the systemic diseases such as sarcoidosis, and may sometimes be the presenting symptom of the disease. In this case report, we present an unusual reaction of the sarcoid granuloma to carbonic anhydrase enzyme inhibitors (CAIs), which was encountered in a patient with ocular sarcoidosis. This observation was taken after a 2-week interval between a CT scan orbits and an MRI orbits which showed a decrease in size from 4×3×4 cm to 2.5×2.5×2 cm, respectiv...

  20. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    Supuran, Claudiu T.

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) × 105 s−1 and kcat/KM ...

  1. Density functional theory study of proton transfer in carbonic anhydrase

    ZHANG Lidong; XIE Daiqian

    2005-01-01

    Proton transfer in carbonic anhydrase II has been studied at the B3LYP/6-31G(D) level. The active site model consists of the zinc ion, four histidine residues, two threonine residues, and three water molecules. Our calculations showed that the proton of the zinc-bound water molecule could be transferred to the nearest water molecule and an intermediate containing H3O+ is then formed. The intermediate is only 1.3 kJ·mol-1 above the reactant complex, whereas the barrier height for the proton transfer is about 8.1 kJ·mol-1.

  2. Quantitative Characterization of the Interaction Space of the Mammalian Carbonic Anhydrase Isoforms I, II, VII, IX, XII, and XIV and their Inhibitors, Using the Proteochemometric Approach.

    Rasti, Behnam; Karimi-Jafari, Mohammad H; Ghasemi, Jahan B

    2016-09-01

    The critical role of carbonic anhydrases in different physiological processes has put this protein family at the center of attention, challenging major diseases like glaucoma, neurological disorders such as epilepsy and Alzheimer's disease, obesity, and cancers. Many QSAR/QSPR (quantitative structure-activity/property relationship) researches have been carried out to design potent carbonic anhydrase inhibitors (CAIs); however, using inhibitors with no selectivity for different isoforms can lead to major side-effects. Given that QSAR/QSPR methods are not capable of covering multiple targets in a unified model, we have applied the proteochemometric approach to model the interaction space that governs selective inhibition of different CA isoforms by some mono-/dihydroxybenzoic acid esters. Internal and external validation methods showed that all models were reliable in terms of both validity and predictivity, whereas Y-scrambling assessed the robustness of the models. To prove the applicability of our models, we showed how structural changes of a ligand can affect the selectivity. Our models provided interesting information that can be useful for designing inhibitors with selective behavior toward isoforms of carbonic anhydrases, aiding in their selective inhibition. PMID:26990115

  3. The effects of some bromophenols on human carbonic anhydrase isoenzymes.

    Taslimi, Parham; Gülçin, İlhami; Öztaşkın, Necla; Çetinkaya, Yasin; Göksu, Süleyman; Alwasel, Saleh H; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrases (CAs, EC 4.2.1.1), which are involved in a variety of physiological and pathological processes, are ubiquitous metalloenzymes mainly catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate ([Formula: see text]) and proton (H(+)). In this study, a dozen of bromophenol derivatives (1-12) were evaluated as metalloenzyme CA (EC 4.2.1.1) inhibitors against the human carbonic anhydrase isoenzymes I and II (hCA I and II). Cytosolic hCA I and II isoenzymes were effectively inhibited by bromophenol derivatives (1-12) with Kis in the low nanomolar range of 1.85 ± 0.58 to 5.04 ± 1.46 nM against hCA I and in the range of 2.01 ± 0.52 to 2.94 ± 1.31 nM against hCA II, respectively. PMID:26133541

  4. Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase

    The first neutron crystal structure of carbonic anhydrase is presented. The structure reveals interesting and unexpected features of the active site that affect catalysis. Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO2 to form HCO3− and H+ using a Zn–hydroxide mechanism. The first part of catalysis involves CO2 hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA

  5. Anion inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Vullo, Daniela; Del Prete, Sonia; De Luca, Viviana; Carginale, Vincenzo; Ferraroni, Marta; Dedeoglu, Nurcan; Osman, Sameh M; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2016-03-01

    The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. Here we report and anion inhibition study of the β-CA, VchCAβ with anions and other small molecules which inhibit metalloenzymes. The best VchCAβ anion inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 54-86μM. Diethyldithiocarbonate was also an effective VchCAβ inhibitor, with an inhibition constant of 0.73mM. The halides, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrate, nitrite, stannate, selenate, tellurate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanide, trithiocarbonate, and fluorosulfonate showed affinity in the low millimolar range, with KIs of 2.3-9.5mM. Identification of selective inhibitors of VchCAβ (over the human CA isoforms) may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzyme. PMID:26853167

  6. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors

    Stefánsson, E; Jensen, P K; Eysteinsson, T;

    1999-01-01

    To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture.......To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture....

  7. Carbonic anhydrase immobilized on hollow fiber membranes using glutaraldehyde activated chitosan for artificial lung applications

    Kimmel, J. D.; Arazawa, D. T.; Ye, S.-H.; Shankarraman, V; Wagner, W. R.; Federspiel, W. J.

    2013-01-01

    Extracorporeal CO2 removal from circulating blood is a promising therapeutic modality for the treatment of acute respiratory failure. The enzyme carbonic anhydrase accelerates CO2 removal within gas exchange devices by locally catalyzing HCO3− into gaseous CO2 within the blood. In this work, we covalently immobilized carbonic anhydrase on the surface of polypropylene hollow fiber membranes using glutaraldehyde activated chitosan tethering to amplify the density of reactive amine functional gr...

  8. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

    Pedersen, Daniella Bach; Koch Jensen, Peter; la Cour, Morten;

    2005-01-01

    Carbonic anhydrase inhibitors (CAIs) increase blood flow in the brain and probably also in the optic nerve and retina. Additionally they elevate the oxygen tension in the optic nerve in the pig. We propose that they also raise the oxygen tension in the retina. We studied the oxygen tension in the...... pig retina and optic nerve before and after dorzolamide injection. Also the retinal vessel diameters during carbonic anhydrase inhibition were studied....

  9. Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum.

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-09-15

    We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO2 hydration: PfCAdom showed a kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), whereas PfCA showed a lower activity compared to PfCAdom, with a kcat of 1.4×10(5)s(-1) and kcat/Km of 5.4×10(6)M(-1)×s(-1). PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 9-68μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with KIs in the range of 0.53-0.97mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action. PMID:27480028

  10. Depletion of the "gamma-type carbonic anhydrase-like" subunits of complex I affects central mitochondrial metabolism in Arabidopsis thaliana.

    Fromm, Steffanie; Göing, Jennifer; Lorenz, Christin; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-01-01

    "Gamma-type carbonic anhydrase-like" (CAL) proteins form part of complex I in plants. Together with "gamma carbonic anhydrase" (CA) proteins they form an extra domain which is attached to the membrane arm of complex I on its matrix exposed side. In Arabidopsis two CAL and three CA proteins are present, termed CAL1, CAL2, CA1, CA2 and CA3. It has been proposed that the carbonic anhydrase domain of complex I is involved in a process mediating efficient recycling of mitochondrial CO2 for photosynthetic carbon fixation which is especially important during growth conditions causing increased photorespiration. Depletion of CAL proteins has been shown to significantly affect plant development and photomorphogenesis. To better understand CAL function in plants we here investigated effects of CAL depletion on the mitochondrial compartment. In mutant lines and cell cultures complex I amount was reduced by 90-95% but levels of complexes III and V were unchanged. At the same time, some of the CA transcripts were less abundant. Proteome analysis of CAL depleted cells revealed significant reduction of complex I subunits as well as proteins associated with photorespiration, but increased amounts of proteins participating in amino acid catabolism and stress response reactions. Developmental delay of the mutants was slightly alleviated if plants were cultivated at high CO2. Profiling of selected metabolites revealed defined changes in intermediates of the citric acid cycle and amino acid catabolism. It is concluded that CAL proteins are essential for complex I assembly and that CAL depletion specifically affects central mitochondrial metabolism. PMID:26482706

  11. Carbonic Anhydrase: An Efficient Enzyme with Possible Global Implications

    Christopher D. Boone

    2013-01-01

    Full Text Available As the global atmospheric emissions of carbon dioxide (CO2 and other greenhouse gases continue to grow to record-setting levels, so do the demands for an efficient and inexpensive carbon sequestration system. Concurrently, the first-world dependence on crude oil and natural gas provokes concerns for long-term availability and emphasizes the need for alternative fuel sources. At the forefront of both of these research areas are a family of enzymes known as the carbonic anhydrases (CAs, which reversibly catalyze the hydration of CO2 into bicarbonate. CAs are among the fastest enzymes known, which have a maximum catalytic efficiency approaching the diffusion limit of 108 M−1s−1. As such, CAs are being utilized in various industrial and research settings to help lower CO2 atmospheric emissions and promote biofuel production. This review will highlight some of the recent accomplishments in these areas along with a discussion on their current limitations.

  12. Sclerostin regulates release of bone mineral by osteocytes by induction of carbonic anhydrase 2.

    Kogawa, Masakazu; Wijenayaka, Asiri R; Ormsby, Renee T; Thomas, Gethin P; Anderson, Paul H; Bonewald, Lynda F; Findlay, David M; Atkins, Gerald J

    2013-12-01

    The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings

  13. Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus

    Muhammad Yousuf Ali

    2015-12-01

    Full Text Available The pH and salinity balance mechanisms of crayfish are controlled by a set of transport-related genes. We identified a set of the genes from the gill transcriptome from a freshwater crayfish Cherax quadricarinatus using the Illumina NGS-sequencing technology. We identified and characterized carbonic anhydrase (CA genes and some other key genes involved in systematic acid-base balance and osmotic/ionic regulation. We also examined expression patterns of some of these genes across different sublethal pH levels [1]. A total of 72,382,710 paired-end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. About 37% of the contigs received significant BLAST hits and 22% were assigned gene ontology terms. These data will assist in further physiological-genomic studies in crayfish.

  14. Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus.

    Ali, Muhammad Yousuf; Pavasovic, Ana; Mather, Peter B; Prentis, Peter J

    2015-12-01

    The pH and salinity balance mechanisms of crayfish are controlled by a set of transport-related genes. We identified a set of the genes from the gill transcriptome from a freshwater crayfish Cherax quadricarinatus using the Illumina NGS-sequencing technology. We identified and characterized carbonic anhydrase (CA) genes and some other key genes involved in systematic acid-base balance and osmotic/ionic regulation. We also examined expression patterns of some of these genes across different sublethal pH levels [1]. A total of 72,382,710 paired-end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. About 37% of the contigs received significant BLAST hits and 22% were assigned gene ontology terms. These data will assist in further physiological-genomic studies in crayfish. PMID:26543880

  15. Expression of carbonic anhydrases IX and XII during mouse embryonic development

    Mannisto Susanna

    2006-05-01

    Full Text Available Abstract Background Of the thirteen active carbonic anhydrase (CA isozymes, CA IX and XII have been linked to carcinogenesis. It has been suggested that these membrane-bound CAs participate in cancer cell invasion, which is facilitated by an acidic tumor cell environment. Since active cell migration is a characteristic feature of embryonic development, we set out to explore whether these isozymes are expressed in mouse embryos of different ages. The studies were focused on organogenesis stage. Results Immunohistochemistry demonstrated that both CA IX and XII are present in several tissues of the developing mouse embryo during organogenesis. Staining for CA IX revealed a relatively wide distribution pattern with moderate signals in the brain, lung, pancreas and liver and weak signals in the kidney and stomach. The expression pattern of CA XII in the embryonic tissues was also relatively broad, although the intensity of immunostaining was weak in most tissues. The CA XII-positive tissues included the brain, where the most prominent staining was seen in the choroid plexus, and the stomach, pancreas, liver and kidney. Conclusion Membrane-bound CA isozymes IX and XII are expressed in various tissues during mouse organogenesis. These enzymes may regulate ion and pH homeostasis within the developing embryo.

  16. Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity.

    Bozdag, Murat; Alafeefy, Ahmed M; Vullo, Daniela; Carta, Fabrizio; Dedeoglu, Nurcan; Al-Tamimi, Abdul-Malek S; Al-Jaber, Nabila A; Scozzafava, Andrea; Supuran, Claudiu T

    2015-12-15

    Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with KIs of 7.6-322nM against hCA I, of 0.06-85.4nM against hCA II; of 6.7-152nM against hCA IX and of 0.49-237nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail. PMID:26639945

  17. Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

    Niederhauser Barbara

    2010-07-01

    Full Text Available Abstract Background The β-carbonic anhydrase (CA, EC 4.2.1.1 enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the β-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. Results The novel β-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of β-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 × 105 s-1 and a kcat/KM of 1.1 × 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM and more potently by sulfamide (KI of 0.15 mM. Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM. Conclusions The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.

  18. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

    Jérôme eDoyen

    2013-01-01

    Full Text Available The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases CAIX and CAXII constitute a robust pHi-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX and LS174Tr cells (inducible knock-down for ca9/ca12 were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2 exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of NHE-1 sensitize cells to radiation-induced cell death. Secondly, the pHi-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50% and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating carbonic anhydrases as an anti-cancer strategy.

  19. Non-destructive measurement of carbonic anhydrase activity and the oxygen isotope composition of soil water

    Jones, Sam; Sauze, Joana; Ogée, Jérôme; Wohl, Steven; Bosc, Alexandre; Wingate, Lisa

    2016-04-01

    Carbonic anhydrases are a group of metalloenzymes that catalyse the hydration of aqueous carbon dioxide (CO2). The expression of carbonic anhydrase by bacteria, archaea and eukarya has been linked to a variety of important biological processes including pH regulation, substrate supply and biomineralisation. As oxygen isotopes are exchanged between CO2 and water during hydration, the presence of carbonic anhydrase in plants and soil organisms also influences the oxygen isotope budget of atmospheric CO2. Leaf and soil water pools have distinct oxygen isotope compositions, owing to differences in pool sizes and evaporation rates, which are imparted on CO2during hydration. These differences in the isotopic signature of CO2 interacting with leaves and soil can be used to partition the contribution of photosynthesis and soil respiration to net terrestrial CO2 exchange. However, this relies on our knowledge of soil carbonic anhydrase activity and currently, the prevalence and function of these enzymes in soils is poorly understood. Isotopic approaches used to estimate soil carbonic anhydrase activity typically involve the inversion of models describing the oxygen isotope composition of CO2 fluxes to solve for the apparent, potentially catalysed, rate of oxygen exchange during hydration. This requires information about the composition of CO2 in isotopic equilibrium with soil water obtained from destructive, depth-resolved soil water sampling. This can represent a significant challenge in data collection given the considerable potential for spatial and temporal variability in the isotopic composition of soil water and limited a priori information with respect to the appropriate sampling resolution and depth. We investigated whether we could circumvent this requirement by constraining carbonic anhydrase activity and the composition of soil water in isotopic equilibrium with CO2 by solving simultaneously the mass balance for two soil CO2 steady states differing only in the

  20. Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis.

    Sumi, Kanij Rukshana; Nou, Ill-Sup; Kho, Kang Hee

    2016-08-22

    Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36-37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis. PMID:27188255

  1. Catecholamine-induced vasoconstriction is sensitive to carbonic anhydrase I activation

    Puscas I.

    2001-01-01

    Full Text Available We studied the relationship between alpha- and beta-adrenergic agonists and the activity of carbonic anhydrase I and II in erythrocyte, clinical and vessel studies. Kinetic studies were performed. Adrenergic agonists increased erythrocyte carbonic anhydrase as follows: adrenaline by 75%, noradrenaline by 68%, isoprenaline by 55%, and orciprenaline by 62%. The kinetic data indicated a non-competitive mechanism of action. In clinical studies carbonic anhydrase I from erythrocytes increased by 87% after noradrenaline administration, by 71% after orciprenaline and by 82% after isoprenaline. The increase in carbonic anhydrase I paralleled the increase in blood pressure. Similar results were obtained in vessel studies on piglet vascular smooth muscle. We believe that adrenergic agonists may have a dual mechanism of action: the first one consists of a catecholamine action on its receptor with the formation of a stimulus-receptor complex. The second mechanism proposed completes the first one. By this second component of the mechanism, the same stimulus directly acts on the carbonic anhydrase I isozyme (that might be functionally coupled with adrenergic receptors, so that its activation ensures an adequate pH for stimulus-receptor coupling for signal transduction into the cell, resulting in vasoconstriction.

  2. Glaucoma and the applications of carbonic anhydrase inhibitors.

    Scozzafava, Andrea; Supuran, Claudiu T

    2014-01-01

    Inhibition of carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the treatment of glaucoma, a disease affecting a large number of people and characterized by an elevated intraocular pressure (IOP). At least three isoforms, CA II, IV and XII are targeted by the sulfonamide inhibitors, some of which are clinically used drugs. Acetazolamide, methazolamide and dichlorophenamide are first generation CA inhibitors (CAIs) still used as systemic drugs for the management of this disease. Dorzolamide and brinzolamide represent the second generation inhibitors, being used topically, as eye drops, with less side effects compared to the first generation drugs. Third generation inhibitors have been developed by using the tail approach, but they did not reach the clinics yet. The most promising such derivatives are the sulfonamides incorporating either tails with nitric oxide releasing moieties or hybrid drugs possessing prostaglandin (PG) F agonist moieties in their molecules. Recently, the dithiocarbamates have also been described as CAIs possessing IOP lowering effects in animal models of glaucoma. CAIs are used alone or in combination with other drugs such as adrenergic agonist/antagonists, or PG analogs, being an important component of the antiglaucoma drugs armamentarium. PMID:24146387

  3. Capsaicin: A Potent Inhibitor of Carbonic Anhydrase Isoenzymes

    Betul Arabaci

    2014-07-01

    Full Text Available Carbonic anhydrase (CA, EC 4.2.1.1 is a zinc containing metalloenzyme that catalyzes the rapid and reversible conversion of carbon dioxide (CO2 and water (H2O into a proton (H+ and bicarbonate (HCO3– ion. On the other hand, capsaicin is the main component in hot chili peppers and is used extensively used in spices, food additives and drugs; it is responsible for their spicy flavor and pungent taste. There are sixteen known CA isoforms in humans. Human CA isoenzymes I, and II (hCA I and hCA II are ubiquitous cytosolic isoforms. In this study, the inhibition properties of capsaicin against the slow cytosolic isoform hCA I, and the ubiquitous and dominant rapid cytosolic isozymes hCA II were studied. Both CA isozymes were inhibited by capsaicin in the micromolar range. This naturally bioactive compound has a Ki of 696.15 µM against hCA I, and of 208.37 µM against hCA II.

  4. N-Nitrosulfonamides: A new chemotype for carbonic anhydrase inhibition.

    Nocentini, Alessio; Vullo, Daniela; Bartolucci, Gianluca; Supuran, Claudiu T

    2016-08-15

    A series of N(1)-substituted aromatic sulfonamides was obtained by applying a selective sulfonamide nitration synthetic strategy leading to Ar-SO2NHNO2 derivatives which were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Two human (h) hCA isoforms, the cytosolic hCA II and the transmembrane hCA IX, in addition to the fungal enzyme from Malassezia globosa, MgCA, were included in the study. Most of the new compounds reported selectively inhibited hCA IX over hCA II and at the same time showed effective MgCA inhibitory properties, with KIs ranging between 0.22 and 8.09μM. The N-nitro sulfonamides are a new chemotype with CA inhibitory effects. As hCA IX was recently validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as potential anti-dandruff agents, the N-nitro sulfonamides may be considered as interesting leads for the design of more efficient compounds targeting fungal enzymes. PMID:27290692

  5. A magnificent enzyme superfamily: carbonic anhydrases, their purification and characterization.

    Ozensoy Guler, Ozen; Capasso, Clemente; Supuran, Claudiu T

    2016-10-01

    In this paper, we reviewed the purification and characterization methods of the α-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (α-, β-, γ-, δ-, ζ- and η-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of α-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the η-CAs, considered for long time an α-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. α-CAs possess important application in medicine (as many human α-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO2 capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action. PMID:26118417

  6. Bacterial carbonic anhydrases as drug targets: towards novel antibiotics ?

    ClaudiuT.Supuran

    2011-07-01

    Full Text Available Carbonic anhydrases (CAs, EC 4.2.1.1 are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the a-, b-, and/or g-CA families. In the last decade, the a-CAs from Neisseria spp. and Helicobacter pylori as well as the b-class enzymes from Escherichia coli, H. pylori, Mycobacterium tuberculosis, Brucella spp., Streptococcus pneumoniae, Salmonella enterica and Haemophilus influenzae have been cloned and characterized in detail. For some of these enzymes the X-ray crystal structures were determined, and in vitro and in vivo inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates reported. Although efficient inhibitors have been reported for many such enzymes, only for Nessseria spp., H. pylori, B. suis and S. pneumoniae enzymes it has been possible to evidence inhibition of bacterial growth in vivo. Thus, bacterial CAs represent promising targets for obtaining antibacterials devoid of the resistance problems of the clinically used such agents but further studies are needed to validate these and other less investigated enzymes as novel drug targets

  7. Electropolymerized carbonic anhydrase immobilization for carbon dioxide capture.

    Merle, Geraldine; Fradette, Sylvie; Madore, Eric; Barralet, Jake E

    2014-06-17

    Biomimetic carbonation carried out with carbonic anhydrase (CA) in CO2-absorbing solutions, such as methyldiethanolamine (MDEA), is one approach that has been developed to accelerate the capture of CO2. However, there are several practical issues, such as high cost and limited enzyme stability, that need to be overcome. In this study, the capacity of CA immobilization on a porous solid support was studied to improve the instability in the tertiary amine solvent. We have shown that a 63% porosity macroporous carbon foam support makes separation and reuse facile and allows for an efficient supply and presentation of CO2 to an aqueous solvent and the enzyme catalytic center. These enzymatic supports conserved 40% of their initial activity after 42 days at 70 °C in an amine solvent, whereas the free enzyme shows no activity after 1 h in the same conditions. In this work, we have overcome the technical barrier associated with the recovery of the biocatalyst after operation, and most of all, these electropolymerized enzymatic supports have shown a remarkable increase of thermal stability in an amine-based CO2 sequestration solvent. PMID:24856780

  8. Quaternary ammonium sulfanilamide: a membrane-impermeant carbonic anhydrase inhibitor

    A novel carbonic anhydrase (CA) inhibitor, quaternary ammonium sulfanilamide (QAS), was tested for potency as a CA inhibitor and for its ability to be excluded from permeating biological membranes. Inhibitor titration plots of QAS vs. pure bovine CA II and CA from the gills of the blue crab, Callinectes sapidus, yielded K/sub i/ values of ∼ 15 μM; thus QAS is a relatively weak but effective CA inhibitor. Permeability of the QAS was directly tested by two independent methods. The inhibitor was excluded from human erythrocytes incubated in 5 mM QAS for 24 h as determined using an 18O-labeled mass spectrometer CA assay for intact cells. Also QAS injected into the hemolymph of C. sapidus (1 or 10 mM) did not cross the basal membrane of the gill. The compound was cleared from the hemolymph by 96 h after injection, and at no time during that period could the QAS be detected in homogenates of gill tissue. Total branchial CA activity was only slightly reduced following the QAS injection. These data indicate that QAS is a CA inhibitor to which biological membranes are impermeable and that can be used in vivo and in vitro in the study of membrane-associated CA

  9. Carbonic anhydrase isozymes Ⅸ and Ⅻ in gastric tumors

    Mari Leppilampi; Juha Saarnio; Tuomo J. Karttunen; Jyrki Kivel(a); Silvia Pastorekov(a); Jaromir Pastorek; Abdul Waheed; William S. Sly; Seppo Parkkila

    2003-01-01

    AIM: To systematically study the expression of carbonic anhydrase (CA) isowmes Ⅸ and Ⅻ in gastric tumors.METHODS: We analyzed a representative series of specimens from non-neoplastic gastric mucosa and from various dysplastic and neoplastic gastric lesions for the expression of CA IX and XII. Immunohistochemical staining was performed using isozyme-specific antibodies and biotinstreptavidin complex method.RESULTS: CA IX was highly expressed in the normal gastric mucosa and remained positive in many gastric tumors. In adenomas, CA IX expression significantly decreased towards the high grade dysplasia. However, the expression resumed back to the normal level in well differentiated adenocarcinomas,while it again declined in carcinomas with less differentiation.In comparison, CA Ⅻ showed no or weak immunoreaction in the normal gastric mucosa and was slightly increased in tumors.CONCLUSION: These results demonstrate that CA Ⅸexpression is sustained in several types of gastric tumors.The variations observed in the CA Ⅸ levels support the concept that gastric adenomas and carcinomas are distinct entities and do not represent progressive steps of a single pathway.

  10. Carbonic anhydrase levels and internal lacunar CO/sub 2/ concentrations in aquatic macrophytes

    Weaver, C.I.

    1979-01-01

    Carbonic anhydrase levels were examined in a variety of aquatic macrophytes from different habitats. In general, carbonic anhydrase levels increased across the habitat gradient such that activities were low in submersed aquatic macrophytes and high in emergent macrophytes with floating-leaved and free-floating plants exhibiting intermediate activities. Internal lacunar CO/sub 2/ concentrations were analyzed in relation to carbonic anhydrase activities. There was no correlation between these two parameters. Internal CO/sub 2/ concentrations ranged from low to high in submersed macrophytes, but were low in floating-leaved and emergent macrophytes. The observed internal CO/sub 2/ concentrations are discussed in relation to the individual morphologies of the plants and the environments in which they occurred.

  11. PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can Efficiently Control the Growth of Several Carbonic Anhydrase IX-Expressing Carcinomas.

    Akocak, Suleyman; Alam, M Raqibul; Shabana, Ahmed M; Sanku, Rajesh Kishore Kumar; Vullo, Daniela; Thompson, Harry; Swenson, Erik R; Supuran, Claudiu T; Ilies, Marc A

    2016-05-26

    A series of aromatic/heterocyclic bis-sulfonamides were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores, coupled with either ethylene glycol oligomeric or polymeric diamines to yield bis-sulfonamides with short or long (polymeric) linkers. Testing of novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity in a detailed structure-activity relationship study. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25) able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment. PMID:27144971

  12. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms.

    Buemi, Maria Rosa; De Luca, Laura; Ferro, Stefania; Bruno, Elvira; Ceruso, Mariangela; Supuran, Claudiu T; Pospíšilová, Klára; Brynda, Jiří; Řezáčová, Pavlína; Gitto, Rosaria

    2015-09-18

    A set of heteroaryl-N-carbonylbenzenesulfonamides has been designed, synthesized, and screened as inhibitors of human carbonic anhydrases (hCAs). The new sulfonamide derivatives were tested against hCA I, hCA II, hCA VII, hCA IX, and hCA XII isoforms using acetazolamide (AAZ, 1) and topiramate (TPM, 2) as reference compounds. Six compounds were low nanomolar inhibitors of tumor-associated hCA IX isoform (Ki values 1500 for compound 5c). Thus, these compounds can offer the opportunity to highlight the interactions preventing the inhibition of hCA VII mainly expressed in central nervous system. Thereby, we used structural and computational techniques to study in depth the interaction with hCAs. In an effort to confirm the inhibitory action we determined crystal structures of five selected heteroaryl-N-carbonylbenzenesulfonamides (4a, 4b, 4e, 5c, and 5e) in complex with hCA II. Moreover, to explore the lack of inhibitory effects of selected compounds (e.g.4b and 5c) we also performed docking studies into hCA VII catalytic site. PMID:26276436

  13. Cloning, expression, purification and sulfonamide inhibition profile of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum.

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-09-01

    We report the cloning, purification and characterization of the full domain of carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, which incorporates 358 amino acid residues (from 181 to 538, in the sequence of this 600 amino acid long protein), called PfCAdom. The enzyme, which belongs to the η-CA class showed the following kinetic parameters: kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), being 13.3 times more effective as a catalyst compared to the truncated form PfCA. PfCAdom is more effective than the human (h) isoform hCA I, being around 50% less effective compared to hCA II, one of the most catalytically efficient enzymes known so far. Intriguingly, the sulfonamides CA inhibitors generally showed much weaker inhibitory activity against PfCAdom compared to PfCA, prompting us to hypothesize that the 69 amino acid residues insertion present in the active site of this η-CA is crucial for the active site architecture. The best sulfonamide inhibitors for PfCAdom were acetazolamide, methazolamide, metanilamide and sulfanilamide, with KIs in the range of 366-808nM. PMID:27485387

  14. Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

    Stams, T.; Y. Chen; Boriack-Sjodin, P. A.; Hurt, J. D.; Liao, J; May, J. A.; Dean, T.; Laipis, P; Silverman, D. N.; Christianson, D. W.

    1998-01-01

    Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some...

  15. Expression and Activity of Carbonic Anhydrase IX Is Associated With Metabolic Dysfunction in MDA-MB-231 Breast Cancer Cells

    Ying LI; Wang, Hai; Oosterwijk, Egbert; Tu, Chingkuang; Shiverick, Kathleen T.; Silverman, David N.; Frost, Susan C.

    2009-01-01

    The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a “triple-negative,” basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density-and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracel...

  16. Human carbonic anhydrase II as a host for piano-stool complexes bearing a sulfonamide anchor.

    Monnard, Fabien W; Heinisch, Tillmann; Nogueira, Elisa S; Schirmer, Tilman; Ward, Thomas R

    2011-08-01

    d(6)-piano-stool complexes bearing an arylsulfonamide anchor display sub-micromolar affinity towards human Carbonic Anhydrase II (hCA II). The 1.3 Å resolution X-ray crystal structure of [(η(6)-C(6)Me(6))Ru(bispy 3)Cl](+)⊂ hCA II highlights the nature of the host-guest interactions. PMID:21706094

  17. Suppression of carbonic anhydrase IX leads to aberrant focal adhesion and decreased invasion of tumor cells

    Radvak, P.; Repic, M.; Svastova, E.; Takacova, M.; Csaderova, L.; Strnad, Hynek; Pastorek, J.; Pastorekova, S.; Kopacek, J.

    2013-01-01

    Roč. 29, č. 3 (2013), s. 1147-1153. ISSN 1021-335X Institutional support: RVO:68378050 Keywords : carbonic anhydrase IX * hypoxia * shRNA silencing * microarray * focal adhesion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.191, year: 2013

  18. Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

    Cannon, Gordon C.; Heinhorst, Sabine; Kerfeld, Cheryl A.

    2010-06-23

    Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO2 concentrations by employing a CO2-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO2-fixing enzyme, RubisCO. The efficiency of CO2 fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO2. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.

  19. Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment

    Závadová, Zuzana; Závada, Jan

    2005-01-01

    Roč. 13, č. 5 (2005), s. 977-982. ISSN 1021-335X R&D Projects: GA ČR(CZ) GA203/02/0405 Institutional research plan: CEZ:AV0Z50520514 Keywords : carbonic anhydrase IX * cell adhesion * microenvironment Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.572, year: 2005

  20. Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment

    Závada, Jan; Závadová, Zuzana

    Praha : Mondial Congress, 2004 - (Witz, I.), s. 198 [International Conference on Tumor Microenvironment:Progrssion, Therapy and Prevention /3./. Praha (CZ), 12.10.2004-16.10.2004] Grant ostatní: Bayer Healthcare (US) nemá číslo Keywords : cancer biology * microenvironmnet * carbonic anhydrase IX Subject RIV: EB - Genetics ; Molecular Biology

  1. Gastric Hyperplasia in Mice With Targeted Disruption of the Carbonic Anhydrase Gene Car9

    Ortova Gut, M.; Parkkila, S.; Vernerová, Z.; Rohde, E.; Závada, Jan; Höcker, M.; Pastorek, J.; Karttunen, T.; Gibadulinová, G.; Závadová, Zuzana; Knobeloch, K. P.; Wiedenmann, B.; Svoboda, Jan; Horak, I.; Pastoreková, S.

    2002-01-01

    Roč. 123, č. 12 (2002), s. 1889-1903. ISSN 0016-5085 R&D Projects: GA ČR GV312/96/K205 Keywords : Carbonic Anhydrases * Knock-aou * Differantiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.440, year: 2002

  2. Gastric hyperplasia in mice with targeted disruption of the carbonic anhydrase gene Car9

    Ortova-Gut, M.; Parkkila, S.; Vernerová, Z.; Rohde, E.; Závada, Jan; Hocker, M.; Pastorek, J.; Karttunen, T.; Gibadulinová, A.; Závadová, Zuzana; Knobeloch, K.-P.; Wiedernmann, B.; Svoboda, Jan; Horak, I.; Pastoreková, S.

    2002-01-01

    Roč. 123, č. 6 (2002), s. 1889-1903. ISSN 0016-5085 R&D Projects: GA ČR GV312/96/K205 Institutional research plan: CEZ:AV0Z5052915 Keywords : mouse carbonic anhydrase Car9 * gastric hyperplasia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.440, year: 2002

  3. Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    De Luca, Viviana; Vullo, Daniela; Del Prete, Sonia; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-02-15

    We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. PMID:26778292

  4. The In vitro Inhibitory Effects of Some Disinfectants on Enzyme Activity of Carbonic Anhydrase from Rainbow Trout (Oncorhynchus Mykiss Gills

    ??kriye Aras Hisar

    2005-01-01

    Full Text Available Traditional treatments of parasitic and bacterial disease in aquaculture are based on chemotherapeutic compounds. Although, a lot of compounds are used on fish treatment, their undesirable effects are not known in detail. In this study, the effects of some disinfectants - malachite green, methylene blue, potassium permanganate, chloramine-T, copper sulphate and formalin - on Rainbow Trout (RT gill Carbonic Anhydrase (CA which plays a key role in gas exchange, acid-base balance, osmoregulation and ionoregulation were investigated in vitro. For this purpose, CA was purified from RT gills by using sepharose-4$-L tyrosine-sulfanylamide affinity gel chromatography method at initial. CA enzyme with 55.56 (EU/mg proteins specific activity was purified with a yield of 40 % and 104.8-fold finally. I (inhibition values of malachite green, 50 methylene blue, potassium permanganate, chloramine-T and copper sulphate were determined as 0.05 mM, 0.023 mM, 0.15 mM, 0.32 mM and 5.39nM by means of activity % [disinfectant] graphs, respectively. In conclusion our data showed that all the disinfectants except formalin had the in vitro inhibitory effects on the rainbow trout gill CA enzyme whose inhibition could be hazardous to physiological functions such as osmoregulation and acid-base balance in fish.

  5. Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

    van Karnebeek, Clara D.; Sly, William S.; Ross, Colin J.; Salvarinova, Ramona; Yaplito-Lee, Joy; Santra, Saikat; Shyr, Casper; Horvath, Gabriella A.; Eydoux, Patrice; Lehman, Anna M.; Bernard, Virginie; Newlove, Theresa; Ukpeh, Henry; Chakrapani, Anupam; Preece, Mary Anne; Ball, Sarah; Pitt, James; Vallance, Hilary D.; Coulter-Mackie, Marion; Nguyen, Hien; Zhang, Lin-Hua; Bhavsar, Amit P.; Sinclair, Graham; Waheed, Abdul; Wasserman, Wyeth W.; Stockler-Ipsiroglu, Sylvia

    2014-01-01

    Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child. PMID:24530203

  6. Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing

    Pedersen, D B; Eysteinsson, T; Stefánsson, E;

    2004-01-01

    Prostaglandins are important in blood flow regulation. Carbon dioxide (CO(2)) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension...... (ONPO(2)), the authors investigated how indomethacin affects ONPO(2) and the ONPO(2) increases caused by CO(2) breathing and carbonic anhydrase inhibition in the pig....

  7. Carbonic Anhydrase and Zinc in Plant Physiology Anhidrasa Carbónica y Zinc en Fisiología Vegetal

    Dalila Jacqueline Escudero-Almanza; Dámaris Leopoldina Ojeda-Barrios; Ofelia Adriana Hernández-Rodríguez; Esteban Sánchez Chávez; Teresita Ruíz-Anchondo; Juan Pedro Sida-Arreola

    2012-01-01

    Carbonic anhydrase (CA) (EC: 2.4.1.1) catalyzes the rapid conversion of carbon dioxide plus water into a proton and the bicarbonate ion (HCO3-) that can be found in prokaryotes and higher organisms; it is represented by four different families. Carbonic anhydrase is a metalloenzyme that requires Zn as a cofactor and is involved in diverse biological processes including pH regulation, CO2 transfer, ionic exchange, respiration, CO2 photosynthetic fixation, and stomatal closure. Therefore, the r...

  8. [Targeting of type IV carbonic anhydrases in Capan-1 human pancreatic duct cells is concomitant of the polarization].

    Mairal, A; Fanjul, M; Hollande, E

    1996-01-01

    Carbonic anhydrases II and IV play an essential role in the synthesis and secretion of HCO3- ions in pancreatic duct cells. Secretion of these ions is regulated by the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. In the present study, the expression of carbonic anhydrases IV and their targeting to plasma membranes were examined during the growth of human pancreatic duct cells in vitro. Human cancerous pancreatic duct cells of Capan-1 cell line which polarize during their growth were used. We show that: a) these cells express carbonic anhydrases IV continuously during growth in culture, and the expression depends on the stage of growth and the conformation of the cells; b) carbonic anhydrases IV are seen in the cytoplasm in non-polarized cells, but become progressively anchored to plasma membranes as the cells polarize, being targeted to the apical membranes of polarized cells; c) the subcellular distribution of carbonic anhydrases IV indicates that these enzymes are synthetized in rough endoplasmic reticulum and then transported towards the plasma membrane using the classical secretory pathway through the Golgi apparatus. The results indicated that targeting of carbonic anhydrases IV in Capan-1 cells is linked to cellular polarization. PMID:8881572

  9. Acetylcholinesterase and carbonic anhydrase inhibitory properties of novel urea and sulfamide derivatives incorporating dopaminergic 2-aminotetralin scaffolds.

    Özgeriş, Bünyamin; Göksu, Süleyman; Polat Köse, Leyla; Gülçin, İlhami; Salmas, Ramin Ekhteiari; Durdagi, Serdar; Tümer, Ferhan; Supuran, Claudiu T

    2016-05-15

    In the present study a series of urea and sulfamide compounds incorporating the tetralin scaffolds were synthesized and evaluated for their acetylcholinesterase (AChE), human carbonic anhydrase (CA, EC 4.2.1.1) isoenzyme I, and II (hCA I and hCA II) inhibitory properties. The urea and their sulfamide analogs were synthesized from the reactions of 2-aminotetralins with N,N-dimethylcarbamoyl chloride and N,N-dimethylsulfamoyl chloride, followed by conversion to the corresponding phenols via O-demethylation with BBr3. The novel urea and sulfamide derivatives were tested for inhibition of hCA I, II and AChE enzymes. These derivatives exhibited excellent inhibitory effects, in the low nanomolar range, with Ki values of 2.61-3.69nM against hCA I, 1.64-2.80nM against hCA II, and in the range of 0.45-1.74nM against AChE. In silico techniques such as, atomistic molecular dynamics (MD) and molecular docking simulations, were used to understand the scenario of the inhibition mechanism upon approaching of the ligands into the active site of the target enzymes. In light of the experimental and computational results, crucial amino acids playing a role in the stabilization of the enzyme-inhibitor adducts were identified. PMID:27068142

  10. Anion and sulfonamide inhibition studies of an α-carbonic anhydrase from the Antarctic hemoglobinless fish Chionodraco hamatus.

    Cincinelli, Alessandra; Martellini, Tania; Vullo, Daniela; Supuran, Claudiu T

    2015-12-01

    An α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from the Antarctic hemoglobinless fish Chionodraco hamatus (icefish). The new enzyme, denominated ChaCA, has a good catalytic activity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the low activity human isoform hCA I, with a kcat of 5.3×10(5) s(-1), and a kcat/Km of 3.7×10(7) M(-1) s(-1). The enzyme was inhibited in the submillimolar range by most inorganic anions (cyanate, thiocyanate, cyanide, bicarbonate, halides), whereas sulfamide, sulfamate, phenylboronic/phenylarsonic acids were micromolar inhibitors, with KIs in the range of 9-77 μM. Many clinically used drugs, such as acetazolamide, methazolamide, dorzolamide, brinzolamide, topiramate and benzolamide were low nanomolar inhibitors, with KIs in the range of 39.1-77.6 nM. As the physiology of CO2/bicarbonate transport or the Root effect in this Antarctic fish are poorly understood at this moment, such inhibition data may give a more detailed insight in the role that CAs play in these phenomena, by the use of inhibitors described here as physiologic tools. PMID:26525863

  11. [Mode of action, clinical profile and relevance of carbonic anhydrase inhibitors in glaucoma therapy].

    Eichhorn, M

    2013-02-01

    Since their introduction the local carbonic anhydrase inhibitors (CAH) dorzolamide and brinzolamide have become well established in the drug therapy of glaucoma. They lower intraocular pressure (IOP) by blocking specifically carbonic anhydrase in the ciliary epithelium and thereby the secretion of aqueous humor. The IOP lowering effect is comparable with that of beta-blockers, but less than that of prostaglandin agonists. Because of their specific mode of action they produce an additive pressure lowering effect with any other glaucoma drug. Therefore they are ideal for being combined with other drugs. In addition, CAH may improve perfusion of the posterior eye. Preliminary results in glaucoma patients under dorzolamide therapy suggesting a reduction in the risk of progression due to enhanced blood flow need further confirmation. PMID:23430679

  12. Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization.

    Fleming, R.E.; Parkkila, S; Parkkila, A K; Rajaniemi, H; Waheed, A; Sly, W S

    1995-01-01

    Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-linked isozyme previously identified on the surface of renal tubular epithelium and certain populations of vascular endothelium. This report identifies the regional, cellular, and subcellular localization of CA IV in the rat gut. Northern blot and RT-PCR analyses demonstrated little CA IV expression in stomach or proximal small intestine, but abundant expression in distal small and large intestine. In contrast, CA II mRNA was abu...

  13. Hemocompatibility Assessment of Carbonic Anhydrase Modified Hollow Fiber Membranes for Artificial Lungs

    Oh, Heung-Il; Ye, Sang-Ho; Johnson, Carl A.; Woolley, Joshua R.; Federspiel, William J.; Wagner, William R.

    2010-01-01

    Hollow fiber membrane (HFM)-based artificial lungs can require a large blood-contacting membrane surface area to provide adequate gas exchange. However, such a large surface area presents significant challenges to hemocompatibility. One method to improve carbon dioxide (CO2) transfer efficiency might be to immobilize carbonic anhydrase (CA) onto the surface of conventional HFMs. By catalyzing the dehydration of bicarbonate in blood, CA has been shown to facilitate diffusion of CO2 toward the ...

  14. Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

    Reibring, Claes-Göran; El Shahawy, Maha; Hallberg, Kristina; Kannius-Janson, Marie; Nilsson, Jeanette; Parkkila, Seppo; Sly, William S; Waheed, Abdul; Linde, Anders; Gritli-Linde, Amel

    2014-01-01

    Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution pa...

  15. Slow reactivation of lyophilized bovine carbonic anhydrase upon dissolution in aqueous solution studied by radiotracer techniques

    Carbonic anhydrase undergoes reversible denaturation upon lyophilization. Full reactivation requires 30-60 min equilibration time following dissolution in aqueous buffers. The recombination of the Zn2+ cofactor with the active site, investigated by 65Zn tracer studies, is clearly shown to account for only the initial stage of the reactivation process and amounts to only a few per cent of the total reactivation. (author)

  16. Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

    Modakh, Joyanta K.; Liu, Yu C.; Machuca, Mayra A.; Supuran, Claudiu T.; Roujeinikova, Anna

    2015-01-01

    Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological ...

  17. Toxic Epidermal Necrolysis Induced by the Topical Carbonic Anhydrase Inhibitors Brinzolamide and Dorzolamide

    Chun, Ji Sun; Yun, Sook Jung; Lee, Jee Bum; Kim, Seong Jin; Won, Young Ho; Lee, Seung Chul

    2008-01-01

    Brinzolamide and dorzolamide are highly specific topical carbonic anhydrase inhibitors (CAIs). They lower intraocular pressure (IOP) by reducing the rate of aqueous humour formation without serious side effects. Although systemic CAIs are the most potent medications for lowering intraocular pressure for conditions with ocular hypertension, many cases with adverse systemic reactions have been reported, including Stevens-Johnson syndrome (SJS) and Toxic epidermal necrolysis (TEN). Here, we repo...

  18. Targeting carbonic anhydrase to treat diabetic retinopathy: Emerging evidences and encouraging results

    Diabetic retinopathy (DR) is the leading cause of vision loss among working-age populations in developed countries. Current treatment options are limited to tight glycemic, blood pressure control and destructive laser surgery. Carbonic anhydrases (CAs) are a group of enzymes involving in the rapid conversion of carbon dioxide to bicarbonate and protons. Emerging evidences reveal CA inhibitors hold the promise for the treatment of DR. This article summarizes encouraging results from clinical and animal studies, and reviews the possible mechanisms.

  19. Targeting carbonic anhydrase to treat diabetic retinopathy: Emerging evidences and encouraging results

    Weiwei, Zhang [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China); Hu, Renming, E-mail: taylorzww@gmail.com [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China)

    2009-12-18

    Diabetic retinopathy (DR) is the leading cause of vision loss among working-age populations in developed countries. Current treatment options are limited to tight glycemic, blood pressure control and destructive laser surgery. Carbonic anhydrases (CAs) are a group of enzymes involving in the rapid conversion of carbon dioxide to bicarbonate and protons. Emerging evidences reveal CA inhibitors hold the promise for the treatment of DR. This article summarizes encouraging results from clinical and animal studies, and reviews the possible mechanisms.

  20. Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides.

    Joyanta K Modak

    Full Text Available Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA, an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

  1. Strong topical steroid, NSAID, and carbonic anhydrase inhibitor cocktail for treatment of cystoid macular edema

    Asahi MG

    2015-12-01

    Full Text Available Masumi G Asahi, Gabriela L Bobarnac Dogaru, Spencer M Onishi, Ron P GallemoreRetina Macula Institute, Torrance, CA, USA Purpose: To report the combination cocktail of strong steroid, non-steroidal anti-inflammatory drug (NSAID, and carbonic anhydrase inhibitor drops for treatment of cystoid macular edema. Methods: This is a retrospective case series of patients with cystoid macular edema managed with a topical combination of strong steroid (difluprednate, NSAID, and carbonic anhydrase inhibitor drops. The patients were followed with optical coherence tomography and fluorescein angiography. Results: In our six cases, resolution of the cystic edema with improvement in visual acuity was achieved with the use of a combination cocktail of drops. Leakage on fluorescein angiography and cystic edema on optical coherence tomography both responded to treatment with the topical cocktail of drops. Conclusion: A topical cocktail of strong steroid, NSAID, and carbonic anhydrase inhibitor drops are effective for managing cystoid macular edema. Further studies comparing this combination with more invasive treatments should be undertaken to determine the efficacy of this cocktail over other treatment options. Keywords: birdshot chorioretinopathy, diabetic macular edema, retinal vein occlusion

  2. Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae.

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-08-15

    Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration-dehydration reaction: CO2+H2O⇋HCO3(-)+H(+). Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO2 hydration, with kcat values ranging between (3.4-8.23)×10(5)s(-1) and kcat/KM of (4.1-7.0)×10(7)M(-1)s(-1). A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values ranging between 44 and 91μM. PMID:27283786

  3. Novel alkalistable α-carbonic anhydrase from the polyextremophilic bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration.

    Faridi, Shazia; Satyanarayana, T

    2016-08-01

    The emissions of CO2 into the atmosphere have been constantly rising due to anthropogenic activities, which have led to global warming and climate change. Among various methods proposed for mitigating CO2 levels in the atmosphere, carbonic anhydrase (CA)-mediated carbon sequestration represents a greener and safer approach to capture and convert it into stable mineral carbonates. Despite the fact that CA is an extremely efficient metalloenzyme that catalyzes the hydration of CO2 (CO2 + H2O ↔ HCO3 (-) + H(+)) with a kcat of ∼10(6) s(-1), a thermostable, and alkalistable CA is desirable for the process to take place efficiently. The purified CA from alkaliphilic, moderately thermophilic, and halotolerant Bacillus halodurans TSLV1 (BhCA) is a homodimeric enzyme with a subunit molecular mass of ~37 kDa with stability in a broad pH range between 6.0 and 11.0. It has a moderate thermostability with a T1/2 of 24.0 ± 1.0 min at 60 °C. Based on the sensitivity of CA to specific inhibitors, BhCA is an α-CA; this has been confirmed by nucleotide/amino acid sequence analysis. This has a unique property of stimulation by SO4 (2-), and it remains unaffected by SO3 (2-), NOx, and most other components present in the flue gas. BhCA is highly efficient in accelerating the mineralization of CO2 as compared to commercial bovine carbonic anhydrase (BCA) and is also efficient in the sequestration of CO2 from the exhaust of petrol driven car, thus, a useful biocatalyst for sequestering CO2 from flue gas. PMID:27102616

  4. Evidence that an internal carbonic anhydrase is present in 5% CO2-grown and air-grown Chlamydomonas

    Inorganic carbon (C/sub i/) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% CO2-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C/sub i/) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO2-grown cells also accumulated some C/sub i/, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high CO2-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO2-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase

  5. Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

    Alvarez Bernardo V

    2013-01-01

    Full Text Available Abstract Background Carbonic anhydrase enzymes (CA catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide, a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. Methods We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14, or heart transplantation (HT, n = 13. CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. Results Expression of atrial and brain natriuretic peptide (ANP and BNP were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. Conclusions These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac

  6. Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets.

    Hanff, Erik; Böhmer, Anke; Zinke, Maximilian; Gambaryan, Stepan; Schwarz, Alexandra; Supuran, Claudiu T; Tsikas, Dimitrios

    2016-07-01

    Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 (-)) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS(15)NO were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). cGMP was determined by LC-MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS(15)NO from (15)N-nitrite. In the presence of L-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASP(S239) were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite. PMID:27129464

  7. Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide.

    Shenghua Huang

    Full Text Available The β-class carbonic anhydrases (β-CAs are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion--a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2-HCO(3(- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

  8. Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide.

    Huang, Shenghua; Hainzl, Tobias; Grundström, Christin; Forsman, Cecilia; Samuelsson, Göran; Sauer-Eriksson, A Elisabeth

    2011-01-01

    The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion--a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3)(-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules. PMID:22162771

  9. Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the β-carbonic anhydrase family enzymes.

    Ogawa, Takahiro; Noguchi, Keiichi; Saito, Masahiko; Nagahata, Yoshiko; Kato, Hiromi; Ohtaki, Akashi; Nakayama, Hiroshi; Dohmae, Naoshi; Matsushita, Yasuhiko; Odaka, Masafumi; Yohda, Masafumi; Nyunoya, Hiroshi; Katayama, Yoko

    2013-03-13

    Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO2 hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS. PMID:23406161

  10. Relationship among Photosys- tem Ⅱ carbonic anhydrase, extrinsic polypeptides and manganese cluster

    2001-01-01

    Effects of Photosystem Ⅱ (PS Ⅱ) extrinsic poly- peptides of oxygen-evolving complex and manganese clusters on PSⅡ carbonic anhydrase (CA) were studied with spinach PSⅡ membranes. The result supported that membrane-bound CA is located in the donor side of PSⅡ. The extrinsic polypeptides played an important role of maintaining CA activity. After removing manganese clusters, oxygen evolution activity was inhibited, but PSⅡ-CA activity was unchanged. It was concluded that CA activity is independent of the presence of manganese clusters, and was not directly correlated with oxygen evolution activity.

  11. Effects of carbonic anhydrase inhibition on ventilation-perfusion matching in the dog lung.

    Swenson, E.R.; Robertson, H T; Hlastala, M P

    1993-01-01

    Lung carbonic anhydrase (CA) permits rapid pH responses when changes in regional ventilation or perfusion alter airway and alveolar PCO2. These pH changes affect airway and vascular resistances and lung compliance to optimize the balance of regional ventilation (VA) and perfusion (Q) in the lung. To test the hypothesis that these or other CA-dependent mechanisms contribute to VA/Q matching, we administered acetazolamide (25 mg/kg intravenously) to six anesthetized and paralyzed dogs and measu...

  12. Dithiocarbamates Strongly Inhibit Carbonic Anhydrases and Show Antiglaucoma Action in Vivo

    Carta, Fabrizio; Aggarwal, Mayank; Maresca, Alfonso; Scozzafava, Andrea; McKenna, Robert; Masini, Emanuela; Supuran, Claudiu T.

    2012-01-01

    A series of dithiocarbamates was prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of 4 human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. X-ray crystal structure of hCA II adduct with morpholine dithiocarbamate ...

  13. Chemical Rescue of Enzymes: Proton Transfer in Mutants of Human Carbonic Anhydrase II

    Maupin, C. Mark; Castillo, Norberto; Taraphder, Srabani; Tu, Chingkuang; McKenna, Robert; Silverman, David N.; Voth, Gregory A.

    2011-01-01

    In human carbonic anhydrase II (HCA II) the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II give a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determi...

  14. A Case With Corneal Edema After Application of Topical Carbonic Anhydrase Inhibitor

    Cumurcu, Tongabay

    2008-01-01

    To report an old patient diagnosed as primary open angel glaucoma (POAG), complicated with irreversible corneal edema after application of topical carbonic anhydrase inhibitor. A 81-year-old man with a previous diagnosis of right and left POAG, of 14-years and 5-years duration respectively, was admitted to our clinic. On ophthalmic examination right eye was absolut glaucoma, and intraocular pressure was measured as 34 mmHg, and visual acuity was 20/200 and intraocular pressure 24 mmHg for the...

  15. Carbonic Anhydrase as Pollution Biomarker: An Ancient Enzyme with a New Use

    Trifone Schettino

    2012-11-01

    Full Text Available The measurement of cellular and sub-cellular responses to chemical contaminants (referred to as biomarkers in living organisms represents a recent tool in environmental monitoring. The review focuses on carbonic anhydrase, a ubiquitous metalloenzyme which plays key roles in a wide variety of physiological processes involving CO2 and HCO3−. In the last decade a number of studies have demonstrated the sensitivity of this enzyme to pollutants such as heavy metals and organic chemicals in both humans and wildlife. The review analyses these studies and discusses the potentiality of this enzyme as novel biomarker in environmental monitoring and assessment.

  16. Design, synthesis, and evaluation of NO-donor containing carbonic anhydrase inhibitors to lower intraocular pressure.

    Huang, Qinhua; Rui, Eugene Y; Cobbs, Morena; Dinh, Dac M; Gukasyan, Hovhannes J; Lafontaine, Jennifer A; Mehta, Saurabh; Patterson, Brian D; Rewolinski, David A; Richardson, Paul F; Edwards, Martin P

    2015-03-26

    The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the alkyl side chain of dorzolamide (1) and brinzolamide (2) has led to the discovery of NO-dorzolamide 3a and NO-brinzolamide 4a, which could lower IOP through two mechanisms: CA inhibition to decrease aqueous humor secretion (reduce inflow) and NO release to increase aqueous humor drainage (increase outflow). Compounds 3a and 4a have shown improved efficacy of lowering IOP in both rabbits and monkeys compared to brinzolamide (2). PMID:25728019

  17. Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands.

    Domigan, L J; Andersson, M; Alberti, K A; Chesler, M; Xu, Q; Johansson, J; Rising, A; Kaplan, D L

    2015-10-01

    Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk

  18. The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives

    Maria Giulia Lionetto; Roberto Caricato; Maria Elena Giordano; Trifone Schettino

    2016-01-01

    Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO2 to HCO3 − and H+. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes n...

  19. Carbonic anhydrase in Tectona grandis: kinetics, stability, isozyme analysis and relationship with photosynthesis.

    Tiwari, Anita; Kumar, Pramod; Chawhaan, Pravin H; Singh, Sanjay; Ansari, S A

    2006-08-01

    Carbonic anhydrase (CA, EC: 4.2.1.1) activity in teak (Tectona grandis L.f.) was studied to determine its characteristics, kinetics and isozyme patterns. We also investigated effects of leaf age, plant age and genotype on CA activity and gas exchange parameters. Carbonic anhydrase extracted from leaves in 12 mM veronal buffer, pH 7.8, had a K(m) for CO(2) of 15.20 mM and a V(max) of 35,448 U mg(-1) chlorophyll min(-1), which values declined by 50 and 70%, respectively, after 1 week of storage at 4 degrees C. A 15% native polyacrylamide gel revealed the absence of CA isozymes in teak, with only a single CA band of 45 kD molecular mass observed across 10 segregating half-sib families and groups of trees ranging in age from 10 to 25 years. Activity remained stable during the first month in storage at 0 degrees C, but gradually declined to 25% of the initial value after 1 year in storage. During the period of active growth (February-May), maximal CA activity was observed in fully expanded and illuminated leaves. Significant variation was observed in CA activity across 10 1-year-old half-sib families and 21 5-year-old half-sib families. There was a positive correlation between CA activity and photosynthetic rate in a population of 10-year-old trees (P teak genotypes. PMID:16651256

  20. New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

    Rogez-Florent, Tiphaine; Meignan, Samuel; Foulon, Catherine; Six, Perrine; Gros, Abigaëlle; Bal-Mahieu, Christine; Supuran, Claudiu T; Scozzafava, Andrea; Frédérick, Raphaël; Masereel, Bernard; Depreux, Patrick; Lansiaux, Amélie; Goossens, Jean-François; Gluszok, Sébastien; Goossens, Laurence

    2013-03-15

    Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1μM CA IX inhibitor. PMID:23168081

  1. Zinc Transfer Kinetics of Metallothioneins and Their Fragmentswith Apo-carbonic Anhydrase

    HUANG, Zhong-Xian; LIU, Fang; ZHENG, Qi; YU, Wen-Hao

    2001-01-01

    Tne zinc transfer reactions from Zn7-MT-I, Zn7-MT-Ⅱ, Zn4α fragment (MT-I) and Zn4-α fragment (MT-Ⅱ) to apo-carbonic anhydrase have been studied. In each reaction, no more than one zinc ion per molecule is involved in metal transfer.Zn7-MT-I and Zn7-MT-Ⅱ donate zinc to apo-carbonic anhydrase and de novo constitute it at a comparable efficiency,while Zn7-MT-Ⅱ exhibits a little faster rate. Surprisingiy,Zinc is released from Zn4-α fragment (MT-Ⅱ) with a much faster rate than from Zn4-α fragment (MT-I), whose rate is close to that of Zn7-MT-I. The reason for the difference is still unknown. Introducing complex compounds into this system may give rise to an effect on the reaction. The transfer from Zn7-MT-Ⅱ in the presence of reduced glutathione shows little difference compare to the control, suggesting that the reduced glutathione is not involved in zinc transfer process. However,glutathione disulfide does accelerate this zinc transfer reaction remarkably, indicating that the oxidative factors contribute to zinc rlease from metallothioneins.

  2. Tracking solvent and protein movement during CO2 release in carbonic anhydrase II crystals.

    Kim, Chae Un; Song, HyoJin; Avvaru, Balendu Sankara; Gruner, Sol M; Park, SangYoun; McKenna, Robert

    2016-05-10

    Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3 (-) Previously, the X-ray crystal structures of CO2-bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo- [T = 0 s (CO2-bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the "slow" release of CO2 Two active site waters, WDW (deep water) and WDW' (this study), replace the vacated space created on CO2 release, and another water, WI (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2' (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away ("out") to pointing toward ("in") active site rotameric conformation. This study provides for the first time, to our knowledge, structural "snapshots" of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO2 is released. Comparison of the holo- and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion. PMID:27114542

  3. Evolution of mammalian carbonic anhydrase loci by tandem duplication: close linkage of Car-1 and Car-2 to the centromere region of chromosome 3 of the mouse

    Eicher, E.M. (Jackson Lab., Bar Harbor, ME); Stern, R.H.; Womack, J.E.; Davisson, M.T.; Roderick, T.H.; Reynolds, S.C.

    1976-01-01

    Electrophoretic variants of two carbonic anhydrase enzymes, CAR-1 (CA I) and CAR-2 (CA II), have been found in the laboratory mouse, Mus musculus. These two loci are closely linked to each other and are located on chromosome 3 near its centromere. The close linkage of Car-1 and Car-2 supports the hypothesis that the present-day carbonic anhydrase loci are the result of tandem duplication of an earlier carbonic anhydrase locus with subsequent divergence. The red blood cells of mice of the subspecies M. m. casteneus have significantly reduced levels of CAR-1 and CAR-2.

  4. Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients

    Závada, Jan; Závadová, Zuzana; Zaťovičová, M.; Hyršl, L.; Kawaciuk, I.

    2003-01-01

    Roč. 89, - (2003), s. 1067-1071. ISSN 0007-0920 R&D Projects: GA ČR GA301/99/0356 Institutional research plan: CEZ:AV0Z5052915 Keywords : carbonic anhydrase IX * tumor antigens * cancer diagnostics Subject RIV: EC - Immunology Impact factor: 3.894, year: 2003

  5. Carbonic anhydrase IX expression in clear cell renal cell carcinomas negatively correlates with the proportion of the granular cell component

    Skapa, P.; Hyršl, L.; Závada, Jan; Soukup, J.; Zámečník, J.

    2008-01-01

    Roč. 26, č. 22 (2008), s. 3811-3812. ISSN 0732-183X Institutional research plan: CEZ:AV0Z40550506 Keywords : carbonic anhydrase IX * CAIX * renal carcinoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 17.157, year: 2008

  6. Evolution of the mammary capillary network and carbonic anhydrase activity throughout lactation and during somatotropin treatment in goats

    Nielsen, Mette Benedicte Olaf; Cvek, Katarina; Dahlborn, Kristina

    2010-01-01

    During the normal course of lactation, mammary metabolic activity and blood flow are closely correlated. Six lactating goats were used in this experiment to test the hypothesis that the capillary network and the capillary enzyme, carbonic anhydrase (CA; EC 4.2.1.1) are important regulatory factors...

  7. In folio study of carbonic anhydrase and Rubisco activities in higher C3 plants using 18O and mass spectrometry

    This document studies the effects of a mild water stress and carbonic anhydrase activity by ethoxyzolamide (EZA) on the diffusion of CO2 in leaves, by 18O labelling of O2 and of CO2 associated to mass spectrometry. (A.B.). 5 refs., 2 figs

  8. Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX

    Takáčová, M.; Holotňáková, T.; Vondráček, Jan; Machala, M.; Pěnčíková, K.; Gradin, K.; Poellinger, L.; Pastorek, J.; Pastoreková, S.; Kopáček, J.

    2009-01-01

    Roč. 419, - (2009), s. 419-425. ISSN 0264-6021 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : AhR * carbonic anhydrase IX * dioxin Subject RIV: BO - Biophysics Impact factor: 5.155, year: 2009

  9. Optimizing lutetium 177-anti-carbonic anhydrase IX radioimmunotherapy in an intraperitoneal clear cell renal cell carcinoma xenograft model

    Muselaers, C.H.J.; Oosterwijk, E.; Bos, D.L.; Oyen, W.J.G.; Mulders, P.F.A.; Boerman, O.C.

    2014-01-01

    A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 (177Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT st

  10. Expression and activity of carbonic anhydrase IX is associated with metabolic dysfunction in MDA-MB-231 breast cancer cells.

    Li, Ying; Wang, H.; Oosterwijk, E.; Tu, C.; Shiverick, K.T.; Silverman, D.N.; Frost, S.C.

    2009-01-01

    The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a "triple-negative," basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydra

  11. Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Harrison, J. L.; Campbell, J. W.; Wiederhold, M. L.; Boyan, B. D.

    1997-01-01

    To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

  12. Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus.

    Ali, Muhammad Yousuf; Pavasovic, Ana; Mather, Peter B; Prentis, Peter J

    2015-06-15

    Changes in water quality parameters such as pH and salinity can have a significant effect on productivity of aquaculture species. Similarly, relative osmotic pressure influences various physiological processes and regulates expression of a number of osmoregulatory genes. Among those, carbonic anhydrase (CA) plays a key role in systemic acid-base balance and ion regulation. Redclaw crayfish (Cherax quadricarinatus) are unique in their ability to thrive in environments with naturally varied pH levels, suggesting unique adaptation to pH stress. To date, however, no studies have focused on identification and characterisation of CA or other osmoregulatory genes in C. quadricarinatus. Here, we analysed the redclaw gill transcriptome and characterized CA genes along with a number of other key osmoregulatory genes that were identified in the transcriptome. We also examined patterns of gene expression of these CA genes when exposed to three pH treatments. In total, 72,382,710 paired end Illumina reads were assembled into 36,128 contigs with an average length of 800bp. Approximately 37% of contigs received significant BLAST hits and 22% were assigned gene ontology terms. Three full length CA isoforms; cytoplasmic CA (ChqCAc), glycosyl-phosphatidylinositol-linked CA (ChqCAg), and β-CA (ChqCA-beta) as well as two partial CA gene sequences were identified. Both partial CA genes showed high similarity to ChqCAg and appeared to be duplicated from the ChqCAg. Full length coding sequences of Na(+)/K(+)-ATPase, V-type H(+)-ATPase, sarcoplasmic Ca(+)-ATPase, arginine kinase, calreticulin and Cl(-) channel protein 2 were also identified. Only the ChqCAc gene showed significant differences in expression across the three pH treatments. These data provide valuable information on the gill expressed CA genes and their expression patterns in freshwater crayfish. Overall our data suggest an important role for the ChqCAc gene in response to changes in pH and in systemic acid-base balance in

  13. Otolith Growth and macular Carbonic Anhydrase Reactivity in larval Fish after Development at simulated Microgravity

    Baur, U.; Hilbig, R.; Anken, R.

    Otolith growth in terms of mineralisation mainly depends on the enzyme carbonic anhydrase (CA). CA is located in specialized, mitochondria-rich macular cells (ionocytes), which are involved in the endolymphatic ion exchange, and the enzyme is responsible for the provision of the pH-value necessary for otolithic calcium carbonate deposition. Since it has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish via a down-regulation of CA reactivity, we were prompted to elucidate whether (simulated) microgravity would possibly yield opposite effects. Therefore, larval siblings of cichlid fish (Oreochromis mossambicus) were housed in a submersed, two-dimensional clinostat (tube) during their development. Subsequently, the "physical capacity" (i.e., size) of the otoliths was measured, CA was histochemically demonstrated in ionocytes, and enzyme reactivity was determined densitometrically. The respective data will be communicated at the meeting. Acknowledgement: This work was financially supported by the German Aerospace Center (DLR) (FKZ: 50 WB 9997).

  14. Xanthates and trithiocarbonates strongly inhibit carbonic anhydrases and show antiglaucoma effects in vivo.

    Carta, Fabrizio; Akdemir, Atilla; Scozzafava, Andrea; Masini, Emanuela; Supuran, Claudiu T

    2013-06-13

    Dithiocarbamates (DTCs) were recently discovered as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. A series of xanthates and a trithiocarbonate, structurally related to the DTCs, were prepared by reaction of alcohols/thiols with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms, hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar xanthate/trithiocarbonate inhibitors targeting these CAs were detected. A docking study of some xanthates within the CA II active site showed that these compounds bind in a similar manner with the dithiocarbamates, coordinating monodentately to the Zn(II) ion from the enzyme active site. Several xanthates showed potent intraocular pressure lowering activity in two animal models of glaucoma via the topical administration. Xanthates and thioxanthates represent two novel, promising classes of CA inhibitors. PMID:23647428

  15. Cadmium-Containing Carbonic Anhydrase CDCA1 in Marine Diatom Thalassiosira weissflogii

    Vincenzo Alterio

    2015-03-01

    Full Text Available The Carbon Concentration Mechanism (CCM allows phytoplakton species to accumulate the dissolved inorganic carbon (DIC necessary for an efficient photosynthesis even under carbon dioxide limitation. In this mechanism of primary importance for diatoms, a key role is played by carbonic anhydrase (CA enzymes which catalyze the reversible hydration of CO2, thus taking part in the acquisition of inorganic carbon for photosynthesis. A novel CA, named CDCA1, has been recently discovered in the marine diatom Thalassiosira weissflogii. CDCA1 is a cambialistic enzyme since it naturally uses Cd2+ as catalytic metal ion, but if necessary can spontaneously exchange Cd2+ to Zn2+. Here, the biochemical and structural features of CDCA1 enzyme will be presented together with its putative biotechnological applications for the detection of metal ions in seawaters.

  16. Carbonic anhydrase mediated carbon dioxide sequestration: promises, challenges and future prospects.

    Yadav, Raju R; Krishnamurthi, Kannan; Mudliar, Sandeep N; Devi, S Saravana; Naoghare, Pravin K; Bafana, Amit; Chakrabarti, Tapan

    2014-06-01

    Anthropogenic activities have substantially increased the level of greenhouse gases (GHGs) in the atmosphere and are contributing significantly to the global warming. Carbon dioxide (CO2 ) is one of the major GHGs which plays a key role in the climate change. Various approaches and methodologies are under investigation to address CO2 capture and sequestration worldwide. Carbonic anhydrase (CA) mediated CO2 sequestration is one of the promising options. Therefore, the present review elaborates recent developments in CA, its immobilization and bioreactor methodologies towards CO2 sequestration using the CA enzyme. The promises and challenges associated with the efficient utilization of CA for CO2 sequestration and scale up from flask to lab-scale bioreactor are critically discussed. Finally, the current review also recommends the possible future needs and directions to utilize CA for CO2 sequestration. PMID:24740638

  17. Heavy metal ion inhibition studies of human, sheep and fish α-carbonic anhydrases.

    Demirdağ, Ramazan; Yerlikaya, Emrah; Şentürk, Murat; Küfrevioğlu, Ö İrfan; Supuran, Claudiu T

    2013-04-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb(2+), Co(2+), Hg(2+), Cd(2+), Zn(2+), Se(2+), Cu(2+), Al(3+) and Mn(3+) showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate α-CAs and is probably due to their binding to His64 or the histidine cluster. PMID:22145795

  18. Conformational effects on the circular dichroism of Human Carbonic Anhydrase II: a multilevel computational study.

    Tatyana G Karabencheva-Christova

    Full Text Available Circular Dichroism (CD spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII, with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.

  19. Carbonic anhydrase enzyme as a potential therapeutic target for experimental trichinellosis.

    Saad, Abeer E; Ashour, Dalia S; Abou Rayia, Dina M; Bedeer, Asmaa E

    2016-06-01

    Trichinellosis is a globally distributed helminthic infection. There is a considerable interest in developing new anti-helminthic drugs affecting all the developmental stages of Trichinella. Acetazolamide (carbonic anhydrase (CA) inhibitor) involves a novel mechanism of action by inhibiting such an essential enzyme for parasite metabolism. This work aimed to study the effect of acetazolamide against different stages of T. spiralis in experimental animals. Mice were divided into three groups: group I: infected and treated with acetazolamide on day 2 post infection (P.I.), group II: infected and treated with acetazolamide on day 12 P.I., and group III: infected non-treated. From each group, small intestine and muscles were removed for histopathological and immunohistochemical studies. Also, total adult and muscle larval count were estimated. We found that acetazolamide was effective in reduction of both adult and muscle larval counts. When given early, the effect was more pronounced on the adults (62.7 %). However, the efficacy of the drug against muscle larvae was increased when given late (63 %). Improvement of the intestinal histopathological changes was observed in all the treated groups. Degeneration of encysted larvae with minimal pathologic changes of infected skeletal muscle was observed in the treated groups. Expression of matrix metalloproteinase-9 showed a statistically significant decrease in the intestinal and muscle tissues in all treated groups as compared to the control group. In conclusion, the present study revealed that acetazolamide, carbonic anhydrase inhibitor, could be a promising drug against both adults and larvae of T. spiralis. PMID:26979731

  20. Kinetics of CO2 exchange with carbonic anhydrase immobilized on fiber membranes in artificial lungs.

    Arazawa, D T; Kimmel, J D; Federspiel, W J

    2015-06-01

    Artificial lung devices comprised of hollow fiber membranes (HFMs) coated with the enzyme carbonic anhydrase (CA), accelerate removal of carbon dioxide (CO2) from blood for the treatment of acute respiratory failure. While previous work demonstrated CA coatings increase HFM CO2 removal by 115 % in phosphate buffered saline (PBS), testing in blood revealed a 36 % increase compared to unmodified HFMs. In this work, we sought to characterize the CO2 mass transport processes within these biocatalytic devices which impede CA coating efficacy and develop approaches towards improving bioactive HFM efficiency. Aminated HFMs were sequentially reacted with glutaraldehyde (GA), chitosan, GA and afterwards incubated with a CA solution, covalently linking CA to the surface. Bioactive CA-HFMs were potted in model gas exchange devices (0.0119 m(2)) and tested for esterase activity and CO2 removal under various flow rates with PBS, whole blood, and solutions containing individual blood components (plasma albumin, red blood cells or free carbonic anhydrase). Results demonstrated that increasing the immobilized enzyme activity did not significantly impact CO2 removal rate, as the diffusional resistance from the liquid boundary layer is the primary impediment to CO2 transport by both unmodified and bioactive HFMs under clinically relevant conditions. Furthermore, endogenous CA within red blood cells competes with HFM immobilized CA to increase CO2 removal. Based on our findings, we propose a bicarbonate/CO2 disequilibrium hypothesis to describe performance of CA-modified devices in both buffer and blood. Improvement in CO2 removal rates using CA-modified devices in blood may be realized by maximizing bicarbonate/CO2 disequilibrium at the fiber surface via strategies such as blood acidification and active mixing within the device. PMID:26032115

  1. The most recently discovered carbonic anhydrase, CA XV, is expressed in the thick ascending limb of Henle and in the collecting ducts of mouse kidney.

    Sina Saari

    Full Text Available BACKGROUND: Carbonic anhydrases (CAs are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla. CONCLUSION/SIGNIFICANCE: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis.

  2. Clues for divergent, polymorphic amyloidogenesis through dissection of amyloid forming steps of bovine carbonic anhydrase and its critical amyloid forming stretch.

    Garg, Dushyant Kumar; Kundu, Bishwajit

    2016-07-01

    Certain amino acid stretches are considered 'critical' to trigger amyloidogenesis in a protein. Synthetic peptides corresponding to these stretches are often used as experimental mimics for studying the amyloidogenesis of their parent protein. Here we provide evidence that such simple extrapolation is misleading. We scrutinized each step of amyloid progression of full length bovine carbonic anhydrase (BCA) and compared it with the amyloidogenic process of its critical peptide stretch 201-227 (PepB). We found that under similar solution conditions amyloidogenesis of BCA followed surface-catalyzed secondary nucleation, whereas, that of PepB followed classical nucleation-dependent pathway. AFM images showed that while BCA formed short, thick and branched fibrils, PepB formed thin, long and unbranched fibrils. Structural information obtained by ATR-FTIR spectroscopy suggested parallel arrangement of intermolecular β-sheet in BCA amyloids in contrast to PepB amyloids which arranged into antiparallel β sheets. Amyloids formed by BCA were unable to seed the fibrillation of PepB and vice versa. Even the intermediates formed during lag phase revealed contrasting FTIR and far UV CD signature, hydrophobicity, morphology and cell cytotoxicity. Thus, we propose that sequences other than critical amyloidogenic stretches may significantly influence the initiation, polymerization and final fibrillar morphology of amyloid forming protein. The results have been discussed in light of primary sequence mediated amyloid polymorphism and its importance in the rational design of amyloid nanomaterials possessing desired physico-chemical properties. PMID:27045222

  3. Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma.

    Alafeefy, Ahmed M; Carta, Fabrizio; Ceruso, Mariangela; Al-Tamimi, Abdul-Malek S; Al-Kahtani, Abdulla A; Supuran, Claudiu T

    2016-03-15

    A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (KIs of 81.0-3084 nM), being highly effective as hCA II (KIs in the range of 0.25-10.8 nM), IX (KIs of 3.7-50.4 nM) and XII (KIs of 0.60-52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated. PMID:26875933

  4. Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties.

    Bozdag, Murat; Alafeefy, Ahmed M; Carta, Fabrizio; Ceruso, Mariangela; Al-Tamimi, Abdul-Malek S; Al-Kahtani, Abdulla A; Alasmary, Fatmah A S; Supuran, Claudiu T

    2016-09-15

    Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (KIs of 28.5-2954nM), being highly effective as hCA II (KIs in the range of 0.62-12.4nM) and XII (KIs of 0.54-7.11nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated. PMID:27396930

  5. Expression and characterization of a recombinant psychrophilic γ-carbonic anhydrase (NcoCA) identified in the genome of the Antarctic cyanobacteria belonging to the genus Nostoc.

    De Luca, Viviana; Del Prete, Sonia; Vullo, Daniela; Carginale, Vincenzo; Di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-10-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO2 hydration/dehydration reversible reaction: CO2 + H2O ⇄ [Formula: see text] + H(+). Living organisms encode for at least six distinct genetic families of such catalyst, the α-, β-, γ-, δ-, ζ- and η-CAs. The main function of the CAs is to quickly process the CO2 derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H(+)) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average -1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a γ-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to α-, β-, γ-classes, as well as a limited stability at moderate temperatures. PMID:26226178

  6. Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.

    de Araujo, Charlotte; Arefeen, Dewan; Tadesse, Yohannes; Long, Benedict M; Price, G Dean; Rowlett, Roger S; Kimber, Matthew S; Espie, George S

    2014-09-01

    Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes. PMID:24907906

  7. The integrative segment of the quail Coturnix coturnix japonica. Occurrence and distribution of carbonic anhydrase and complex carbohydrates.

    Gabriella, M G; Menghi, G

    1994-01-01

    As part of a more extensive study into the involvement of carbonic anhydrase in avian excretory function, the occurrence and distribution of this enzyme was investigated in the quail integrative segment. The integrative segment represents, in birds, that part of the intestinal tract where ureteral urine undergoes postrenal modification to form definitive urine. To define the structural peculiarities within the intestinal epithelium, the constituent parts, namely cloaca, rectum and caecum, as ...

  8. Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts

    Ditte, Zuzana; Ditte, Peter; Labudova, Martina; Simko, Veronika; Iuliano, Filippo; Zatovicova, Miriam; Csaderova, Lucia; Pastorekova, Silvia; Pastorek, Jaromir

    2014-01-01

    Background Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA I...

  9. Carbon-13 nuclear magnetic resonance as a probe of side chain orientation and mobility in carboxymethylated human carbonic anhydrase B

    Schoot Uiterkamp, Antonius J.M.; Armitage, Ian M.; Prestegard, James H.; Slomski, John; Coleman, Joseph E.

    1978-01-01

    13C NMR spectra of [1-13C]- and [2-13C]carboxymethyl His-200 human carbonic anhydrase B have been obtained as a function of pH and in the presence and absence of the active site Zn(II) or Cd(II) ion. Chemical shifts of the 1-13C show that the carboxyl is sensitive to two ionization processes, with a

  10. Effects of carbonyl sulfide (COS) and carbonic anhydrase on stomatal conductance

    Yakir, D.; Stimler, K.; Berry, J. A.

    2011-12-01

    The potential use of COS as tracer of the gross, one-way, CO2 flux into plants is based on its co-diffusion with CO2 into leaves without outflux stimulated research on COS-CO2 interactions during leaf gas exchange. We carried out gas exchange measurements of COS and CO2 in 22 plant species representing deciduous and evergreen trees, grasses, and shrubs, under a range of light intensities and ambient COS concentrations, using mid IR laser spectroscopy. A narrow range in the normalized ratio of the net uptake rates of COS (As) and CO2 (Ac; As/Ac*[CO2]/[COS]) was observed, with a mean value of 1.61±0.26. These results reflect the dominance of stomatal conductance over both COS and CO2 uptake, imposing a relatively constant ratio between the two fluxes (except under low light conditions when CO2, but not COS, metabolism is light limited). A relatively constant ratio under common ambient conditions will facilitate the application of COS as a tracer of gross photosynthesis from leaf to global scales. However, its effect on stomatal conductance may require a special attention. Increasing COS concentrations between 250 and 2800 pmol mol-1 (enveloping atmospheric levels) seems to stimulate stomatal conductance. We examined the stimulation of conductance by COS in a range of species and show that there is a large variation with some species showing almost no response while others are highly responsive (up to doubling stomatal conductance). Using C3 and C4 plants with antisense lines abolishing carbonic anhydrase activity, we show that the activity of this enzyme is essential for both the uptake of COS and the enhancement of stomatal conductance by COS. Since carbonic anhydrase catalyzes the conversion of COS to CO2 and H2S it seems likely that the stomata are responding to H2S produced in the mesophyll. In all natural species examined the uptake of COS and CO2 were highly correlated, and there was no relationship between the sensitivity of stomata and the rate of COS uptake

  11. The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932-2015).

    Buzás, György M; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Puşcaş used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Puşcaş for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend. PMID:26108882

  12. Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa

    Carbonic anhydrase (CA) isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated

  13. Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa

    Saarnio Juha

    2005-04-01

    Full Text Available Abstract Background Carbonic anhydrase (CA isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. Methods Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. Results The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. Conclusion The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated.

  14. On the role of carbonic anhydrase in the early phase of fish otolith mineralization

    Beier, M.; Anken, R.

    2006-01-01

    The first step in the formation of fish otoliths, calcified structures which are responsible for the internalization of gravitational information, is based on the action of so-called Tether- (T-) cells. These T-cells appear during the very early development of the inner ear and persist only a few hours. They are characterized by a kinocilium, which is in contrast to the kinocilium of the later developing sensory hair cells not mechanosensory, but binds seeding particles containing glycogen, thereby localizing otolith formation (otolith seeding). Beating cilia distributed throughout the ear agitate seeding particles, thereby inhibiting premature agglutination. In the later development, a protein matrix is formed and mineralization/crystallization takes place. Since the enzyme carbonic anhydrase (CAH) plays a prominent role in otolith mineralization (it provides carbonate for CaCO3 precipitation), we were prompted to investigate histochemically using larval cichlid fish (Oreochromis mossambicus), whether CAH might be present as early as T-cells. Indeed, CAH was present in T-cells with prominent amounts of reaction product being located along the kinocilia and around the seeding particles. These results strongly indicate that kinocilia of T-cells act as structural guides for CAH/bicarbonate transportation towards the early otoliths’ calcification sites. Besides its role in calcification, CAH in the very early stage of otolith seeding may moreover aid in the accretion process of the precursor particles.

  15. Synthesis and Evaluation of New Phthalazine Urea and Thiourea Derivatives as Carbonic Anhydrase Inhibitors

    Nurcan Berber

    2013-01-01

    Full Text Available A new series of phthalazine substituted urea and thiourea derivatives were synthesized, and their inhibitory effects on the activity of purified human carbonic anhydrases (hCAs I and II were evaluated. 2H-Indazolo[2,1-b]phthalazine-trione derivative (1 was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and nitro group was reduced to amine derivative (2 with SnCl2·2H2O. The compound was reacted with isocyanates and isothiocyanates to get the final products (3a–p. The results showed that all the synthesized compounds inhibited the CA isoenzymes activity. 3a (IC50 = 6.40 µM for hCA I and 6.13 µM for hCA II has the most inhibitory effect. The synthesized compounds are very bulky to be able to bind near the zinc ion, and they much more probably bind as the coumarin derivatives.

  16. Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization.

    Fei, Xiaoyao; Chen, Shaoyun; Liu, Dai; Huang, Chunjie; Zhang, Yongchun

    2016-09-01

    Two functionalized SBA-15 [amine-functionalized SBA-15 (AFS) and epoxy-functionalized SBA-15 (GFS)] with different types of functional groups were synthesized by a hydrothermal process and post functionalized with 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS), respectively. They were used for the immobilization of carbonic anhydrase (CA). The physicochemical properties of the functionalized SBA-15 were characterized by X-ray powder diffraction (XRD), N2 adsorption-desorption, (13)C, (29)Si solid-state nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). Before and after CA was immobilized on AFS and GFS, the effects of temperature and pH value on the enzyme activity, storage stability, and reusability were investigated using para-nitrophenyl acetate (p-NPA) assay. CA/GFS showed a better performance with respect to storage stability and reusability than CA/AFS. Moreover, the amount of CaCO3 precipitated over CA/AFS was less than that precipitated over CA/GFS, which was almost equal to that precipitated over the free CA. The results indicate that the epoxy group is a more suitable functional group for covalent bonding with CA than the amino group, and GFS is a promising support for CA immobilization. PMID:27215831

  17. Acetylcholinesterase and carbonic anhydrase isoenzymes I and II inhibition profiles of taxifolin.

    Gocer, Hulya; Topal, Fevzi; Topal, Meryem; Küçük, Murat; Teke, Dilek; Gülçin, İlhami; Alwasel, Saleh H; Supuran, Claudiu T

    2016-06-01

    Taxifolin, also known as dihydroquercetin, is a flavonoid commonly found in plants. Carbonic anhydrase (CA, EC 4.2.1.1) plays an important role in many critical physiological events including carbon dioxide (CO2)/bicarbonate ([Formula: see text]) respiration and pH regulation. There are 16 known CA isoforms in humans, of which human hCA isoenzymes I and II (hCA I and II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of taxifolin against the slow cytosolic isoenzyme hCA I, and the ubiquitous and dominant rapid cytosolic isoenzyme hCA II were studied. Taxifolin, as a naturally bioactive flavonoid, has a Ki of 29.2 nM against hCA I, and 24.2 nM against hCA II. For acetylcholinesterase enzyme (AChE) inhibition, Ki parameter of taxifolin was determined to be 16.7 nM. These results clearly show that taxifolin inhibited both CA isoenzymes and AChE at the nM levels. PMID:25893707

  18. Enzyme renaturation to higher activity driven by the sol-gel transition: Carbonic anhydrase

    Vinogradov, Vladimir V.; Avnir, David

    2015-09-01

    We describe a so-far unknown route for renaturing denatured enzymes, namely subjecting the denatured enzyme to an oxide sol-gel transition. The phenomenon was revealed in a detailed study of denatured carbonic anhydrase which was subjected to an alumina sol-gel transition, up to the thermally stabilizing entrapment in the final xerogel. Remarkably, not only that the killed enzyme regained its activity during the sol-gel process, but its activity increased to 180% of the native enzyme. To the best of our knowledge, this is the first report of enhanced activity following by renaturing (a “Phoenix effect”). Kinetic study which revealed a five-orders of magnitude (!) increase in the Arrhenius prefactor upon entrapment compared to solution. Circular dichroism analysis, differential scanning calorimetry, zeta potential analyses as well as synchronous fluorescence measurements, all of which were used to characterize the phenomenon, are consistent with a proposed mechanism which is based on the specific orienting interactions of the active site of the enzyme with respect to the alumina interface and its pores network.

  19. Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase.

    Bozdag, Murat; Carta, Fabrizio; Vullo, Daniela; Isik, Semra; AlOthman, Zeid; Osman, Sameh M; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-01

    Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, were investigated for the inhibition of α- and β-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the Saccharomyces cerevisiae β-CA, scCA. The yeast and its β-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA I inhibitors (K(I)s of 66.5-910 nM), were more effective as hCA II inhibitors (K(I)s of 8.9-107 nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, with inhibition constants ranging between 6.4 and 259 nM. The detailed structure activity relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms. PMID:25669351

  20. Innovative molecular diagnosis of Trichinella species based on β-carbonic anhydrase genomic sequence.

    Zolfaghari Emameh, Reza; Kuuslahti, Marianne; Näreaho, Anu; Sukura, Antti; Parkkila, Seppo

    2016-03-01

    Trichinellosis is a helminthic infection where different species of Trichinella nematodes are the causative agents. Several molecular assays have been designed to aid diagnostics of trichinellosis. These assays are mostly complex and expensive. The genomes of Trichinella species contain certain parasite-specific genes, which can be detected by polymerase chain reaction (PCR) methods. We selected β-carbonic anhydrase (β-CA) gene as a target, because it is present in many parasites genomes but absent in vertebrates. We developed a novel β-CA gene-based method for detection of Trichinella larvae in biological samples. We first identified a β-CA protein sequence from Trichinella spiralis by bioinformatic tools using β-CAs from Caenorhabditis elegans and Drosophila melanogaster. Thereafter, 16 sets of designed primers were tested to detect β-CA genomic sequences from three species of Trichinella, including T. spiralis, Trichinella pseudospiralis and Trichinella nativa. Among all 16 sets of designed primers, the primer set No. 2 efficiently amplified β-CA genomic sequences from T. spiralis, T. pseudospiralis and T. nativa without any false-positive amplicons from other parasite samples including Toxoplasma gondii, Toxocara cati and Parascaris equorum. This robust and straightforward method could be useful for meat inspection in slaughterhouses, quality control by food authorities and medical laboratories. PMID:26639312

  1. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-01-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions. PMID:27385052

  2. Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Ferraroni, Marta; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-03-01

    The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2-87.0nM. Other compounds, with medium potency against VchCAβ, (KIs in the range of 275-463nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (KIs in the range of 4.51-8.57μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes. PMID:26850377

  3. Mitochondrial gamma carbonic anhydrases are required for complex I assembly and plant reproductive development.

    Fromm, Steffanie; Braun, Hans-Peter; Peterhansel, Christoph

    2016-07-01

    Complex I of the mitochondrial electron transport chain (mETC) in plants contains an extra domain that is made up from proteins homologous to prokaryotic gamma-carbonic anhydrases (γCA). This domain has been suggested to participate in complex I assembly or to support transport of mitochondrial CO2 to the chloroplast. Here, we generated mutants lacking CA1 and CA2 - two out of three CA proteins in Arabidopsis thaliana. Double mutants were characterized at the developmental and physiological levels. Furthermore, the composition and activity of the mETC were determined, and mutated CA versions were used for complementation assays. Embryo development of double mutants was strongly delayed and seed development stopped before maturation. Mutant plants could only be rescued on sucrose media, showed severe stress symptoms and never produced viable seeds. By contrast, callus cultures were only slightly affected in growth. Complex I was undetectable in the double mutants, but complex II and complex IV were upregulated concomitant with increased oxygen consumption in mitochondrial respiration. Ectopic expression of inactive CA variants was sufficient to complement the mutant phenotype. Data indicate that CA proteins are structurally required for complex I assembly and that reproductive development is dependent on the presence of complex I. PMID:26889912

  4. SYNTHESIS AND EVALUATION OF NEW PHTHALAZINE SUBSTITUTED β-LACTAM DERIVATIVES AS CARBONIC ANHYDRASE INHIBITORS.

    Berber, Nurcan; Arslan, Mustafa; Bilen, Çiğdem; Sackes, Zübeyde; Gençer, Nahit; Arslan, Oktay

    2015-01-01

    A new series of phthalazine substituted β-lactam derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated. 2H-Indazolo[2,1-b]phthala- zine-trione derivative was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and the nitro group was reduced to 13-(4-aminophenyl)-3,3-dimethyl-3,4-dihydro- 2H-indazolo[1,2-b]phthalazine-1,6,11(13H)-trione with SnCl2 · 2H2O. The reduced compound was re- acted with different aromatic aldehydes, and phthalazine substituted imines were synthesized. The imine compounds undergo (2+2) cycloaddition reactions with ketenes to produce 2H-indazolo[2,1-b]phthala-zine-trione substituted β-lactam derivatives. The β-lactam compounds were tested as inhibitors of the CA isoenzyme activity. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. 1-(4-(3,3-dimethyl- 1,6,1 1-trioxo-2,3,4,6,11,13-hexahydro-1H-indazolo[1,2-b]phthalazin-13- yl)phenyl)-2-oxo-4-p-tolylazetidin-3-yl acetate (IC50 = 6.97 µM for hCA I and 8.48 µM for hCA II) had the most inhibitory effect. PMID:26615643

  5. Indazole, Pyrazole, and Oxazole Derivatives Targeting Nitric Oxide Synthases and Carbonic Anhydrases.

    Maccallini, Cristina; Di Matteo, Mauro; Vullo, Daniela; Ammazzalorso, Alessandra; Carradori, Simone; De Filippis, Barbara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Supuran, Claudiu T; Amoroso, Rosa

    2016-08-19

    Nitric oxide (NO) is an essential endogenous mediator with a physiological role in the central nervous system as neurotransmitter and neuromodulator. A growing number of studies have demonstrated that abnormal nitrergic signaling is a crucial event in the development of neurodegeneration. In particular, the uncontrolled production of NO by neuronal nitric oxide synthase (nNOS) is observed in several neurodegenerative diseases. Moreover, it is well recognized that specific isoforms of human carbonic anhydrase (hCA) physiologically modulate crucial pathways of signal processing and that low expression of CA affects cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive impairments. In light of this, dual agents that are able to target both NOS (inhibition) and CA (activation) could be useful drug candidates for the treatment of Alzheimer's disease, aging, and other neurodegenerative diseases. In the present work, we show the design, synthesis, and in vitro biological evaluation of new nitrogen-based heterocyclic compounds. Among the tested molecules, 2-amino-3-(4-hydroxyphenyl)-N-(1H-indazol-5-yl)propanamide hydrochloride (10 b) was revealed to be a potent dual agent, able to act as a selective nNOS inhibitor and activator of the hCA I isoform. PMID:27377568

  6. [Immobilization and characterization of carbonic anhydrase on the surface of hollow fiber membrane of polymethyl pentene].

    Wang, Qinmei; Zhang, Dihua; Zhang, Jingxia

    2009-07-01

    We immobilized carbonic anhydrase (CA) onto the surface of membrane oxygenator of polymethyl pentene (PMP) to enhance the removal of carbon dioxide in blood by two steps. We first introduced hydroxyl groups onto PMP surface by water plasma treatment, and then coupled CA onto PMP surface by using cyanate bromide (CNBr) as a crosslinker. After plasma treatment, the contact angle with water and chemical composition of PMP surface were characterized by analysis system of surface contact angle and XPS. Using p-nitrophenyl acetate (p-NPA) as a substrate, the activity, concentration, storage stability and re-usability of immobilized CA on PMP hollow fibers were studied by ultraviolet spectrophotometer. The preliminary data showed that hydroxyl groups could be introduced on the surface of PMP by water plasma treatment, and CA with catalysis activity could be successfully introduced onto PMP surface in high immobilization efficiency. The activity of covalently immobilized CA increased with the increase of concentration of CNBr, and the maximum was 73% of the theoretical activity of CA spread on PMP surface in monolayer in studied range. Covalently immobilized CA showed higher reusability compared to physically adsorbed CA, and higher storage stability compared to free CA in solution at 37 degrees C. The method would be used potentially in the membrane oxygenator to improve the capacity of removal of carbon dioxide in blood in the future. PMID:19835148

  7. Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase--a new reductase.

    Jing, Qing; Okrasa, Krzysztof; Kazlauskas, Romas J

    2009-01-01

    One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions. PMID:19115310

  8. Sulfamate inhibitor S4 influences carbonic anhydrase IX ectodomain shedding in colorectal carcinoma cells.

    Hektoen, Helga Helseth; Ree, Anne Hansen; Redalen, Kathrine Røe; Flatmark, Kjersti

    2016-10-01

    Carbonic anhydrase IX (CAIX) is a pivotal pH regulator under hypoxia, which by its tumor-specific expression represents an attractive target for cancer therapy. Here, we report on effects of the sulfamate CAIX inhibitor S4 (4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate) in colorectal carcinoma cell lines. S4 was administered under experimental hypoxia or normoxia to HT29, KM20L2 and HCT116 cells. Effects on survival, proliferation, pH, lactate extrusion and CAIX protein expression were evaluated. S4 treatment resulted in attenuated hypoxia-induced extracellular acidification and reduced clonogenic survival under hypoxia in HT29 cells. The pH effects were present only in a [Formula: see text]-free buffer system and were accompanied by decreased lactate extrusion. The main finding of this work was that S4 treatment caused alterations in CAIX ectodomain shedding. This merits further investigation to understand how sulfamates influence CAIX activity and how such drugs may be of use in cancer treatment. PMID:26244271

  9. The role of carbonic anhydrase in C4 photosynthesis

    Studer, Anthony [Life Sciences Research Foundation, Baltimore, MD (United States)

    2015-10-01

    Current pressures on the global food supply have accelerated the urgency for a second green revolution using novel and sustainable approaches to increase crop yield and efficiency. This proposal outlines experiments to address fundamental questions regarding the biology of C4 photosynthesis, the method of carbon fixation utilized by the most productive food, feed and bioenergy crops. Carbonic anhydrase (CA) has been implicated in multiple cellular functions including nitrogen metabolism, water use efficiency, and photosynthesis. CA catalyzes the first dedicated step in C4 photosynthesis, the hydration of CO2 into bicarbonate, and is potentially rate limiting in C4 grasses. Using insertional mutagenesis, we have generated CA mutants in maize, and propose the characterization of these mutants using phenotypic, physiological, and transcriptomic profiling to assay the plant’s response to altered CA activity. In addition, florescent protein tagging experiments will be employed to study the subcellular localization of CA paralogs, providing critical data for modeling carbon fixation in C4 plants. Finally, I propose parallel experiments in Setaria viridis to explore its relevance as model C4 grass. Using a multifaceted approach, this proposal addresses important questions in basic biology, as well as the need for translation research in response to looming global food challenges.

  10. Carbonic anhydrase XII expression is associated with histologic grade of cervical cancer and superior radiotherapy outcome

    Lee Su-Kyoung

    2010-11-01

    Full Text Available Abstract Background To investigate whether expression of carbonic anhydrase XII (CA12 is associated with histologic grade of the tumors and radiotherapy outcomes of the patients with invasive cervical cancer. Methods CA12 expression was examined by immunohistochemical stains in cervical cancer tissues from 183 radiotherapy patients. Histological grading was classified as well (WD, moderately (MD or poorly differentiated (PD. Oligonucleotide microarray experiment was performed using seven cervical cancer samples to examine differentially expressed genes between WD and PD cervical cancers. The association between CA12 and histological grade was analyzed by chi-square test. CA12 and histological grades were analyzed individually and as combined CA12 and histologic grade categories for effects on survival outcome. Results Immunohistochemical expression of CA12 was highly associated with the histologic grade of cervical cancer. Lack of CA12 expression was associated with PD histology, with an odds ratio of 3.9 (P = 0.01. Microarray analysis showed a fourfold reduction in CA12 gene expression in PD tumors. CA12 expression was marginally associated with superior disease-free survival. Application of the new combined categories resulted in further discrimination of the prognosis of patients with moderate and poorly differentiated tumor grade. Conclusions Our study indicates that CA12 may be used as a novel prognostic marker in combination with histologic grade of the tumors.

  11. Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II.

    Taraphder, Srabani; Maupin, C Mark; Swanson, Jessica M J; Voth, Gregory A

    2016-08-25

    The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II. PMID:27063577

  12. Characterization of carbonic anhydrase II from Chlorella vulgaris in bio-CO2 capture.

    Li, Li; Fu, Ming-Lai; Zhao, Yong-Hao; Zhu, Yun-Tian

    2012-11-01

    Carbonic anhydrase II (CA II) can catalyze the reversible hydration reaction of CO(2) at a maximum of 1.4 × 10(6) molecules of CO(2) per second. The crude intracellular enzyme extract containing CA II was derived from Chlorella vulgaris. A successful CO(2) capture experiment with the presence of calcium had been conducted on the premise that the temperature was conditioned at a scope of 30-40 °C, that the biocatalyst-nurtured algal growth period lasted 3 days, and that pH ranged from7.5 to 8.5. Ions of K(+), Na(+), Ca(2+), Co(2+), Cu(2+), Fe(3+), Mg(2+), Mn(2+), and Zn(2+) at 0.01, 0.1, and 0.5 M were found to exhibit no more than 30 % inhibition on the residual activity of the biocatalyst. It is reasonable to expect that calcification catalyzed by microalgae presents an alternative to geological carbon capture and sequestration through a chain of fundamental researches carried on under the guidance of sequestration technology. PMID:22821342

  13. Carbonic anhydrase XII expression is associated with histologic grade of cervical cancer and superior radiotherapy outcome

    To investigate whether expression of carbonic anhydrase XII (CA12) is associated with histologic grade of the tumors and radiotherapy outcomes of the patients with invasive cervical cancer. CA12 expression was examined by immunohistochemical stains in cervical cancer tissues from 183 radiotherapy patients. Histological grading was classified as well (WD), moderately (MD) or poorly differentiated (PD). Oligonucleotide microarray experiment was performed using seven cervical cancer samples to examine differentially expressed genes between WD and PD cervical cancers. The association between CA12 and histological grade was analyzed by chi-square test. CA12 and histological grades were analyzed individually and as combined CA12 and histologic grade categories for effects on survival outcome. Immunohistochemical expression of CA12 was highly associated with the histologic grade of cervical cancer. Lack of CA12 expression was associated with PD histology, with an odds ratio of 3.9 (P = 0.01). Microarray analysis showed a fourfold reduction in CA12 gene expression in PD tumors. CA12 expression was marginally associated with superior disease-free survival. Application of the new combined categories resulted in further discrimination of the prognosis of patients with moderate and poorly differentiated tumor grade. Our study indicates that CA12 may be used as a novel prognostic marker in combination with histologic grade of the tumors

  14. Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases.

    Pinard, Melissa A; Boone, Christopher D; Rife, Brittany D; Supuran, Claudiu T; McKenna, Robert

    2013-11-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I-XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity. PMID:24090602

  15. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune.

    Vullo, Daniela; De Luca, Viviana; Del Prete, Sonia; Carginale, Vincenzo; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2015-04-15

    A carbonic anhydrase (CA, EC 4.2.1.1) belonging to the γ-class has been cloned, purified and characterized from the Antarctic cyanobacterium Nostoc commune. The enzyme showed a good catalytic activity for the physiologic reaction (hydration of carbon dioxide to bicarbonate and a proton) with the following kinetic parameters, kcat of 9.5×10(5)s(-1) and kcat/KM of 8.3×10(7)M(-1)s(-1), being the γ-CA with the highest catalytic activity described so far. A range of aromatic/heterocyclic sulfonamides and one sulfamate were investigated as inhibitors of the new enzyme, denominated here NcoCA. The best NcoCA inhibitors were some sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, acetazolamide, benzolamide, dorzolamide, brinzolamide and topiramate, which showed inhibition constants in the range of 40.3-92.3nM. As 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and γ-CAs are closely associated in carboxysomes of cyanobacteria for enhancing the affinity of RubisCO for CO2 and the efficiency of photosynthesis, investigation of this new enzyme and its affinity for modulators of its activity may bring new insights in these crucial processes. PMID:25773015

  16. Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii.

    Vullo, Daniela; Del Prete, Sonia; Osman, Sameh M; De Luca, Viviana; Scozzafava, Andrea; Alothman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2014-01-01

    The δ-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a δ-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with KIs of 49.6-118nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (KIs of 375-897nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with KIs of 4.27-9.20μM. The inhibition profile of the δ-CA is very different from that of α-, β- and γ-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the δ-CA active site. PMID:24314394

  17. Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum.

    Vullo, Daniela; Del Prete, Sonia; Fisher, Gillian M; Andrews, Katherine T; Poulsen, Sally-Ann; Capasso, Clemente; Supuran, Claudiu T

    2015-02-01

    The η-carbonic anhydrases (CAs, EC 4.2.1.1) were recently discovered as the sixth genetic class of this metalloenzyme superfamily, and are so far known only in protozoa, including various Plasmodium species, the causative agents of malaria. We report here an inhibition study of the η-CA from Plasmodium falciparum (PfCA) against a panel of sulfonamides and one sulfamate compound, some of which are clinically used. The strongest inhibitors identified were ethoxzolamide and sulthiame, with KIs of 131-132 nM, followed by acetazolamide, methazolamide and hydrochlorothiazide (KIs of 153-198 nM). Brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM. An interesting observation was that the more efficient PfCA inhibitors are representative of several scaffolds and chemical classes, including benzene sulfonamides, monocyclic/bicyclic heterocyclic sulfonamides and compounds with a more complex scaffold (i.e., the sugar sulfamate derivative, topiramate, and the coxibs, celecoxib and valdecoxib). A comprehensive inhibition study of small molecules for η-CAs is needed as a first step towards assessing PfCA as a druggable target. The present work identifies the first known η-CA inhibitors and provides a platform for the development of next generation novel PfCA inhibitors. PMID:25533402

  18. Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor.

    Wang, Cun; Hu, Honghong; Qin, Xue; Zeise, Brian; Xu, Danyun; Rappel, Wouter-Jan; Boron, Walter F; Schroeder, Julian I

    2016-02-01

    Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 (-) enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels. PMID:26764375

  19. Prediction of binding modes and affinities of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide inhibitors to the carbonic anhydrase receptor by docking and ONIOM calculations.

    Samanta, Pabitra Narayan; Das, Kalyan Kumar

    2016-01-01

    Inhibition activities of a series of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamides against the human carbonic anhydrase II (HCAII) enzyme have been explored by employing molecular docking and hybrid QM/MM methods. The docking protocol has been employed to assess the best pose of each ligand in the active site cavity of the enzyme, and probe the interactions with the amino acid residues. The docking calculations reveal that the inhibitor binds to the catalytic Zn(2+) site through the deprotonated sulfonamide nitrogen atom by making several hydrophobic and hydrogen bond interactions with the side chain residues depending on the substituted moiety. A cross-docking approach has been adopted prior to the hybrid QM/MM calculation to validate the docked poses. A correlation between the experimental dissociation constants and the docked free energies for the enzyme-inhibitor complexes has been established. Two-layered ONIOM calculations based on QM/MM approach have been performed to evaluate the binding efficacy of the inhibitors. The inhibitor potency has been predicted from the computed binding energies after taking into account of the electronic phenomena associated with enzyme-inhibitor interactions. Both the hybrid (B3LYP) and meta-hybrid (M06-2X) functionals are used for the description of the QM region. To improve the correlation between the experimental biological activity and the theoretical results, a three-layered ONIOM calculation has been carried out and verified for some of the selected inhibitors. The charge transfer stabilization energies are calculated via natural bond orbital analysis to recognize the donor-acceptor interaction in the binding pocket of the enzyme. The nature of binding between the inhibitors and HCAII active site is further analyzed from the electron density distribution maps. PMID:26619075

  20. Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma.

    Vullo, Daniela; Durante, Mariaconcetta; Di Leva, Francesco Saverio; Cosconati, Sandro; Masini, Emanuela; Scozzafava, Andrea; Novellino, Ettore; Supuran, Claudiu T; Carta, Fabrizio

    2016-06-23

    A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines and COS as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates, the xanthates, and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the pharmacologically relevant human (h) hCAs isoforms I, II, IX, and XII in vitro and showed KIs spanning between the low and medium nanomolar range. By means of a computational study, the MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering effects and showed interesting activity. PMID:27253845

  1. Self-healing of early age cracks in cement-based materials by mineralization of carbonic anhydrase microorganism

    Qian, Chunxiang; Chen, Huaicheng; Ren, Lifu; Luo, Mian

    2015-01-01

    This research investigated the self-healing potential of early age cracks in cement-based materials incorporating the bacteria which can produce carbonic anhydrase. Cement-based materials specimens were pre-cracked at the age of 7, 14, 28, 60 days to study the repair ability influenced by cracking time, the width of cracks were between 0.1 and 1.0 mm to study the healing rate influenced by width of cracks. The experimental results indicated that the bacteria showed excellent repairing ability...

  2. Membrane Specific Carbonic Anhydrase (CA-IV) Expression in Bovine Lung: The Effects of Alcoholic and Non-Alcoholic Drinks

    DEMİR, Nazan; NADAROĞLU, Hayrunnisa

    2002-01-01

    Carbonic anhydrase (CA) (carbonate hydrolyase: E. C. 4.2.1.1) from bovine lung was purified by a new method and characterized. The purification level was 4306-fold. The optimum temperature for maximum enzyme activity was 37.5°C. The optimum pH was 7.4, varying between 3.5 and 10.0. SDS-polyacryamide gel electrophoresis (3-10% discontinuous SDS-PAGE) showed two distinct bands for CA-IV. The molecular weights of the enzymes were found to be approximately 54.000 and 29.000, respectively. ...

  3. Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis

    Yu, Su-jong; Yoon, Jung-Hwan; Lee, Jeong-Hoon; Myung, Sun-jung; Jang, Eun-sun; Kwak, Min-Sun; Cho, Eun-Ju; Jang, Ja-June; Kim, Yoon-jun; Lee, Hyo-Suk

    2011-01-01

    Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth wa...

  4. Immunocytochemical localization of carbonic anhydrase in the pseudobranch tissue of the rainbow trout Oncorhynchus mykiss

    S. M. RAHIM; A. G. MAZLAN; K. D. SIMON; J. P. DELAUNOY; P. LAURENT

    2014-01-01

    本文题目:虹鳟假鳃组织中的碳酸酐酶免疫细胞化学定位Immunocytochemical localization of carbonic anhydrase in the pseudobranch tissue of the rainbow trout Oncorhynchus mykiss研究目的:假腮的功能早已引起科学家兴趣,但还有待阐明。本文通过研究硬骨鱼类品种虹鳟鱼(Oncorhynchus mykiss)的假腮碳酸酐酶的免疫定位,来探讨假腮碳酸酐酶的生理功能。研究方法:免疫组织化学染色技术。重要结论:免疫组化结果显示碳酸酐酶分布在假腮细胞中,更精确地说是在其细胞顶端分布。细胞基底端、管状系统、毛细血管和柱细胞均无免疫染色。免疫细胞化学定位进一步验证了这些结果,并显示一部分是细胞质碳酸酐酶,其余的与细胞膜结构连接。此外,腔隙层未显示出免疫过氧化物酶的活性。本研究揭示了假腮碳酸酐酶的功能与细胞外介质有关,碳酸酐酶能干预传入神经纤维刺激机制。%Pseudobranch function has long interested scientists, but its role has yet to be elucidated. Several studies have suggested that pseudobranchs serve respiratory, osmoregulatory, and sensory functions. This work investigated the immunolocalization of pseudobranch carbonic anhydrase (CA) in the teleost fish species rainbow trout (Oncor-hynchus mykiss) to clarify its physiological function. CA was purified from rainbow trout gil s O. mykiss and specific antibodies were raised. Immunoblotting between tissue homogenates of pseudobranch and gil CA antibodies showed specific immunostaining with only one band corresponding to CA in the pseudobranch homogenate. Results of im-munohistochemical technique revealed that CA was distributed within pseudobranch cells and more precisely in the apical parts (anti-vascular) of cells. The basal (vascular) parts of cells, tubular system, blood capillaries, and pillar cells were not immunostained. Immunocytochemistry confirmed these results and

  5. Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

    Mayank Aggarwal

    2014-03-01

    Full Text Available Human carbonic anhydrases (CAs are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type of a variant of CA II in which His64 is replaced with Ala (H64A CA II can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations the activity of H64A CA II.

  6. Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

    Amaya Blanco-Rivero

    Full Text Available BACKGROUND: Cah3 is the only carbonic anhydrase (CA isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2 conditions. RESULTS/CONCLUSIONS: In the present work we demonstrate that after transfer to low CO(2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. SIGNIFICANCE: This is the first report of a CA being post

  7. External carbonic anhydrase in three Caribbean corals: quantification of activity and role in CO2 uptake

    Tansik, Anna L.; Fitt, William K.; Hopkinson, Brian M.

    2015-09-01

    Scleractinian corals have complicated inorganic carbon ( C i) transport pathways to support both photosynthesis, by their symbiotic dinoflagellates, and calcification. The first step in C i acquisition, uptake into the coral, is critical as the diffusive boundary layer limits the supply of CO2 to the surface and HCO3 - uptake is energy intensive. An external carbonic anhydrase (eCA) on the oral surface of corals is thought to facilitate CO2 uptake by converting HCO3 - into CO2, helping to overcome the limitation imposed by the boundary layer. However, this enzyme has not yet been identified or detected in corals, nor has its activity been quantified. We have developed a method to quantify eCA activity using a reaction-diffusion model to analyze data on 18O removal from labeled C i. Applying this technique to three species of Caribbean corals ( Orbicella faveolata, Porites astreoides, and Siderastrea radians) showed that all species have eCA and that the potential rates of CO2 generation by eCA greatly exceed photosynthetic rates. This demonstrates that eCA activity is sufficient to support its hypothesized role in CO2 supply. Inhibition of eCA severely reduces net photosynthesis in all species (on average by 46 ± 27 %), implying that CO2 generated by eCA is a major carbon source for photosynthesis. Because of the high permeability of membranes to CO2, CO2 uptake is likely driven by a concentration gradient across the cytoplasmic membrane. The ubiquity of eCA in corals from diverse genera and environments suggests that it is fundamental for photosynthetic CO2 supply.

  8. Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

    Rudenko, Natalia N; Ignatova, Lyudmila K; Ivanov, Boris N

    2007-01-01

    Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids. PMID:17347907

  9. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration.

    Fan, Li-Hai; Liu, Ning; Yu, Ming-Rui; Yang, Shang-Tian; Chen, Huan-Lin

    2011-12-01

    Carbonic anhydrase (CA) has recently gained renewed interests for its potential as a mass-transfer facilitator for CO(2) sequestration. However, the low stability and high price severely limit its applications. In this work, the expression of α-CA from Helicobacter pylori on the outer membrane of Escherichia coli using a surface-anchoring system derived from ice nucleation protein (INP) from Pseudomonas syringae was developed. To find the best surface anchoring motif, full-length INP (114 kDa), truncated INP (INP-NC, 33 kDa), and INP's N-domain with first two subunits (INP-N, 22 kDa) were evaluated. Two vectors, pKK223-3 and pET22b(+), with different promoters (T7 and Tac) were used to construct the fusion genes, and for each vector, three recombinant strains, each expressing a different length of the fusion protein, were obtained. SDS-PAGE, Western blot, immunofluorescence microscopy, FACS, and whole-cell ELISA confirmed the expression of fusion proteins on the surface of E. coli. The smallest fusion protein with INP-N as the anchoring motif had the highest expression level and CA activity, suggesting that INP-N is the best carrying protein due to its smaller size. Also, the T7 promoter in pET22b(+) induced with 0.2 mM IPTG gave high protein expression levels, whereas the Tac promoter in pKK223-3 gave low expression levels. The surface displayed CA was at least twofold more stable than that of the free form, and did not show any adverse effect on cell growth and outer membrane integrity. Cells with surface displayed CA were successfully used to facilitate CO(2) sequestration in contained liquid membrane (CLM). PMID:21732326

  10. Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos.

    Karakostis, Konstantinos; Costa, Caterina; Zito, Francesca; Brümmer, Franz; Matranga, Valeria

    2016-06-01

    Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus. PMID:27230618

  11. Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

    Claes-Göran Reibring

    Full Text Available Carbonic anhydrases (CAs play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for

  12. Histochemical localisation of carbonic anhydrase in the inner ear of developing cichlid fish, Oreochromis mossambicus

    Beier, M.; Hilbig, R.; Anken, R.

    2008-12-01

    Inner ear otolith growth in terms of mineralisation mainly depends on the enzyme carbonic anhydrase (CAH). CAH is located in specialised, mitochondria-rich macular cells (ionocytes), which are involved in the endolymphatic ion exchange, and the enzyme is responsible for the provision of the pH-value necessary for otolithic calcium carbonate deposition. In the present study, for the first time the localisation of histochemically demonstrated CAH was analysed during the early larval development of a teleost, the cichlid fish Oreochromis mossambicus. CAH-reactivity was observed already in stage 7 animals (onset of otocyst development; staging follows Anken et al. [Anken, R., Kappel, T., Slenzka, K., Rahmann, H. The early morphogenetic development of the cichlid fish, Oreochromis mossambicus (Perciformes, Teleostei). Zool. Anz. 231, 1-10, 1993]). Neuroblasts (from which sensory and supporting cells are derived) proved to be CAH-positive. Already at stage 12 (hatch), CAH-positive regions could be attributed to ionocyte containing regions both in the so-called meshwork and patches area of the macula (i.e., clearly before ionocytes can be identified on ultrastructural level or by employing immunocytochemistry). In contrast to the circumstances observed in mammalian species, sensory hair cells stained negative for CAH in the cichlid. With the onset of stage 16 (finray primordia in dorsal fin, yolk-sac being increasingly absorbed), CAH-reactivity was observed in the vestibular nerve. This indicates the onset of myelinisation and thus commencement of operation. The localisation of CAH in the inner ear of fish (especially the differences in comparison to mammals) is discussed on the basis of its role in otolith calcification. Since the vestibular system is a detector of acceleration and thus gravity, also aspects regarding effects of altered gravity on CAH and hence on the mineralisation of otoliths in an adaptive process are addressed.

  13. Ethylene bis-imidazoles are highly potent and selective activators for isozymes VA and VII of carbonic anhydrase, with a potential nootropic effect

    Draghici, Bogdan; Vullo, Daniela; Akocak, Suleyman; Walker, Ellen A; Supuran, Claudiu T.; Ilies, Marc A.

    2014-01-01

    A series of ethylene bis-imidazoles was synthesized via a novel microwave-mediated synthesis. Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against CA VA and CA VII, also displaying excellent selectivity against other CA isozymes present in this organ.

  14. Sites of calcium uptake of fish otoliths correspond with macular regions rich of carbonic anhydrase

    Beier, M.; Anken, R.; Hilbig, R.

    2006-01-01

    Based on pharmacological data, it has been suggested that the enzyme carbonic anhydrase (CAH) plays a prominent role in the mineralization of fish otoliths. To directly test this proposal, the topographical distribution of CAH was histochemically analyzed in the utricular and saccular maculae of larval cichlid fish Oreochromis mossambicus. Further investigations were focussed on the sites of otolithic calcium uptake using the fluorescent calcium tracer alizarin-complexone (AC). Both in the utricle and the saccule, CAH-reactivity was prominent in regions on both sides of the sensory macula (centrifugal (cf) and centripetal (cp) areas), which reportedly contain ionocytes, specialized cells regulating the ionic composition of the endolymph. (The terms centrifugal and centripetal were chosen instead of lateral and medial, because the saccule is positioned perpendicular to the utricle; “lateral” and “medial” thus do not allow an unambiguous allocation of the respective regions.) In the saccule, the size of cf and cp did not differ from each other, whereas, in the utricle, cp was considerably larger as compared to cf (CAH-reactivity per μm2 was nearly identical in both areas of both endorgans). AC-incubation resulted in a fluorescent band on the proximal surface of the otoliths (this surface lies next to the sensory epithelium). In saccular otoliths (sagittae), the area of the band did not differ between centrifugal and centripetal otolith regions, whereas in the utricular otoliths (lapilli), the area of the centripetal AC-band was larger in size as compared to the centrifugal one (AC-fluorescence per μm2 did not differ between the areas analyzed in both types of otoliths). These results strongly suggest that calcium/carbonate uptake of otoliths takes place especially in those regions of their proximal face which are located adjacent to CAH-rich areas of the macular epithelium. It is thus concluded that CAH is directly involved in otolith calcification. The

  15. Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

    Lerman Michael I

    2009-03-01

    Full Text Available Abstract Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1. Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1 HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2 There is no evidence of

  16. Linking Carbonic Anhydrase Abundance and Diversity in Soils to Ecological Function

    Pang, E.; Meredith, L. K.; Welander, P. V.

    2015-12-01

    Carbonic anhydrase (CA) is an ancient enzyme widespread among bacteria, archaea, and eukarya that catalyzes the following reaction: CO2 + H2O ⇌ HCO3- + H+. Its functions are critical for key cellular processes such as concentrating CO2 for autotrophic growth, pH regulation, and pathogen survival in hosts. Currently, there are six known CA classes (α, β, γ, δ, η, ζ) arising from several distinct evolutionary lineages. CA are widespread in sequenced genomes, with many organisms containing multiple classes of CA or multiple CA of the same class. Soils host rich microbial communities with diverse and important ecological functions, but the diversity and abundance of CA in soils has not been explored. CA appears to play an important, but poorly understood, role in some biogeochemical cycles such as those of CO2 and its oxygen isotope composition and also carbonyl sulfide (COS), which are potential tracers in predictive carbon cycle models. Recognizing the prevalence and functional significance of CA in soils, we used a combined bioinformatics and molecular biology approach to address fundamental questions regarding the abundance, diversity, and function of CA in soils. To characterize the abundance and diversity of the different CA classes in soils, we analyzed existing soil metagenomic and metatranscriptomic data from the DOE Joint Genome Institute databases. Out of the six classes of CA, we only found the α, β, and γ classes to be present in soils, with the β class being the most abundant. We also looked at genomes of sequenced soil microorganisms to learn what combination of CA classes they contain, from which we can begin to predict the physiological role of CA. To characterize the functional roles of the different CA classes in soils, we collected soil samples from a variety of biomes with diverse chemical and physical properties and quantified the rate of two CA-mediated processes: soil uptake of COS and acceleration of the oxygen isotope exchange

  17. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  18. Carbonic anhydrase activators: X-ray crystal structure of the adduct of human isozyme II with L-histidine as a platform for the design of stronger activators.

    Temperini, Claudia; Scozzafava, Andrea; Puccetti, Luca; Supuran, Claudiu T

    2005-12-01

    Activation of the carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, and IV with l-histidine and some of its derivatives has been investigated by kinetic and X-ray crystallographic methods. l-His was a potent activator of isozymes I and IV (activation constants in the range of 4-33microM), and a moderate hCA II activator (activation constant of 113microM). Both carboxy- as well as amino-substituted l-His derivatives, such as the methyl ester or the dipeptide carnosine (beta-Ala-His), acted as more efficient activators as compared to l-His. The X-ray crystallographic structure of the hCA II-l-His adduct showed the activator to be anchored at the entrance of the active site cavity, participating in an extended network of hydrogen bonds with the amino acid residues His64, Asn67, and Gln92 and, with three water molecules connecting it to the zinc-bound water. Although the binding site of l-His is similar to that of histamine, the first CA activator for which the X-ray crystal structure has been reported in complex with hCA II (Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C. T. Biochemistry1997, 36, 10384) there are important differences of binding between the two structurally related activators, since histamine interacts among others with Asn67 and Gln92 (similarly to l-His), but also with Asn62 and not His64, whereas the number of water molecules connecting them to the zinc-bound water is different (two for histamine, three for l-His). Furthermore, the imidazole moieties of the two activators adopt different conformations when bound to the enzyme active site. Since neither the amino- nor carboxy moieties of l-His participate in interactions with amino acid moieties of the active site, they can be derivatized for obtaining more potent activators, with pharmacological applications for the enhancement of synaptic efficacy. This may constitute a novel approach for the treatment of Alzheimer's disease, aging, and other conditions in

  19. Formation of local native-like tertiary structures in the slow refolding reaction of human carbonic anhydrase II as monitored by circular dichroism on tryptophan mutants.

    Andersson, D; Freskgård, P O; Jonsson, B H; Carlsson, U

    1997-04-15

    In the present study, near-UV CD kinetic measurements on mutants, in which one Trp residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCAII). In addition, the formation of the active site was probed by the binding of a fluorescent sulfonamide inhibitor. The development of the individual Trp CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCAII at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Trp residues 16, 97, and 245 were analyzed. Trp16 probes the N-terminal domain (amino acid residues 1-25), and this part is forming its tertiary structure slower than the major domain (amino acid residues 26-260) of the protein molecule, which contains the active site and a dominating beta-sheet. An essentially native structure of the major domain seems to act as a template for the correct folding of the N terminus. Trp97 is located in a hydrophobic cluster comprising beta-strands 3-5 in the protein core. Previously, we have shown that this region is remarkably stable and compact, and stopped-flow fluorescence data indicate that Trp97 is buried in an apolar compact cluster within a few milliseconds [Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Martensson, L.-G., Gentile, M., Bóren, K., & Carlsson, U. (1995) Biochemistry 34, 8606-8620; Jonasson, P., Aronsson, G., Carlsson, U., & Jonsson, B.-H. (1997) Biochemistry 36 (in press)]. Here it is shown that the development of the native tertiary structure at Trp97 occurs in the minute time domain. Trp245 is located in a long loop between the N-terminal domain and the core structure. Although this Trp has attained native-like fluorescence properties within the dead time of the CD experiment, it assumes a

  20. Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas.

    Lounnas, Nadia; Rosilio, Célia; Nebout, Marielle; Mary, Didier; Griessinger, Emmanuel; Neffati, Zouhour; Chiche, Johanna; Spits, Hergen; Hagenbeek, Thijs J; Asnafi, Vahid; Poulsen, Sally-Ann; Supuran, Claudiu T; Peyron, Jean-François; Imbert, Véronique

    2013-06-01

    The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN-/- mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL. PMID:23348702

  1. Novel sulfonamide bearing coumarin scaffolds as selective inhibitors of tumor associated carbonic anhydrase isoforms IX and XII.

    Chandak, Navneet; Ceruso, Mariangela; Supuran, Claudiu T; Sharma, Pawan K

    2016-07-01

    Four novel scaffolds consisting of total 24 compounds (1a-1o, 2a-2c, 3a-3c and 4a-4c) bearing aromatic sulfonamide and coumarin moieties connected through various linkers were synthesized in order to synergize the inhibition potential of both the moieties against four selected human carbonic anhydrase isoforms (hCA I, II, IX & XII). All compounds were found to be potent inhibitors of tumor associated hCA IX & XII while at the same time required large amounts to inhibit off-targeted housekeeping hCA I & II. Selectivity was more pronounced against hCA II over I, and hCA XII over IX. Results were compared with antitumor drug acetazolamide. One derivative 2b of series 2 was found to be a better selective inhibitor of hCA IX and XII. PMID:27137360

  2. Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors.

    Ceruso, Mariangela; Antel, Sabrina; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-01

    New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with KIs in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents. PMID:25792500

  3. Identifying motor and sensory myelinated axons in rabbit peripheral nerves by histochemical staining for carbonic anhydrase and cholinesterase activities

    Riley, Danny A.; Sanger, James R.; Matloub, Hani S.; Yousif, N. John; Bain, James L. W.

    1988-01-01

    Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myeliated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50 percent or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.

  4. Influence of Carbonic Anhydrase Activity in Terrestrial Vegetation on the 18O Content of Atmospheric CO2

    Gillon, Jim; Yakir, Dan

    2001-03-01

    The oxygen-18 (18O) content of atmospheric carbon dioxide (CO2) is an important indicator of CO2 uptake on land. It has generally been assumed that during photosynthesis, oxygen in CO2 reaches isotopic equilibrium with oxygen in 18O-enriched water in leaves. We show, however, large differences in the activity of carbonic anhydrase (which catalyzes CO2 hydration and 18O exchange in leaves) among major plant groups that cause variations in the extent of 18O equilibrium (θeq). A clear distinction in θeq between C3 trees and shrubs, and C4 grasses makes atmospheric C18OO a potentially sensitive indicator to changes in C3 and C4 productivity. We estimate a global mean θeq value of ~0.8, which reasonably reconciles inconsistencies between 18O budgets of atmospheric O2 (Dole effect) and CO2.

  5. DNA cloning, characterization, and inhibition studies of an α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Del Prete, Sonia; Isik, Semra; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Scozzafava, Andrea; Supuran, Claudiu T; Capasso, Clemente

    2012-12-13

    We have cloned, purified, and characterized an α-carbonic anhydrase (CA, EC 4.2.1.1) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity, and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which are methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (KI values in the range 0.69-8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered until now. PMID:23181552

  6. The extremo-α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium azorense is highly inhibited by sulfonamides.

    Vullo, Daniela; De Luca, Viviana; Scozzafava, Andrea; Carginale, Vincenzo; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2013-08-01

    The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) is the most effective CA known to date. Here we investigated the inhibition profile of this enzyme with a series of aromatic and heterocyclic sulfonamides, and one sulfamate. Many clinically used sulfonamides, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, celecoxib and sulpiride were low nanomolar/subnanomolar SazCA inhibitors (KIs in the range of 0.9-10.8 nM) whereas simple aromatic derivatives were less effective as SazCA inhibitors. The inhibition profile of SazCA is slightly different from that of the related enzyme from S. yellostonense (SspCA), investigated earlier by our groups. PMID:23777827

  7. A systematic quantitative approach to rational drug design and discovery of novel human carbonic anhydrase IX inhibitors.

    Sethi, Kalyan K; Verma, Saurabh M

    2014-08-01

    Drug design involves the design of small molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were performed for a series of carbonic anhydrase IX inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques with the help of SYBYL 7.1 software. The large set of 36 different aromatic/heterocyclic sulfamates carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, such as hCA IX, was chosen for this study. The conventional ligand-based 3D-QSAR studies were performed based on the low energy conformations employing database alignment rule. The ligand-based model gave q(2) values 0.802 and 0.829 and r(2) values 1.000 and 0.994 for CoMFA and CoMSIA, respectively, and the predictive ability of the model was validated. The predicted r(2) values are 0.999 and 0.502 for CoMFA and CoMSIA, respectively. SEA (steric, electrostatic, hydrogen bond acceptor) of CoMSIA has the significant contribution for the model development. The docking of inhibitors into hCA IX active site using Glide XP (Schrödinger) software revealed the vital interactions and binding conformation of the inhibitors. The CoMFA and CoMSIA field contour maps are well in agreement with the structural characteristics of the binding pocket of hCA IX active site, which suggests that the information rendered by 3D-QSAR models and the docking interactions can provide guidelines for the development of improved hCA IX inhibitors as leads for various types of metastatic cancers including those of cervical, renal, breast and head and neck origin. PMID:24090419

  8. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene. PMID:27119954

  9. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    Adnan Shah

    Full Text Available Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954 and gca (cg0155 genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.

  10. Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes

    Orito Kensuke

    2011-03-01

    Full Text Available Abstract Background Carbonic anhydrase (CA of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes. Methods Chicken CA-II was purified by a combination of column chromatography. The levels of CA-II in the hemolysate of the chicken were determined using the ELISA system in blood samples from 279 female chickens, ages 1 to 93 weeks, 69 male chickens, ages 3 to 59 weeks and 52 weeks female Araucana-chickens. Results The mean concentration of CA-II in hemolysate from 1-week-old female was 50.8 ± 11.9 mg/g of Hb. The mean levels of CA-II in 25-week-old (188.1 ± 82.6 mg/g of Hb, 31-week-old (193.6 ± 69.7 mg/g of Hb and 49-week-old (203.8 ± 123.5 mg/g of Hb female-chickens showed the highest level of CA-II. The levels of CA-II in female WL-chickens significantly decreased at 63 week (139.0 ± 19.3 mg/g of Hb. The levels of CA-II in female WL-chicken did not change from week 63 until week 93.The mean level of CA-II in hemolysate of 3-week-old male WL-chickens was 78.3 ± 20.7 mg/g of Hb. The levels of CA-II in male WL-chickens did not show changes in the week 3 to week 59 timeframe. The mean level of CA-II in 53-week-old female Araucana-chickens was 23.4 ± 1.78 mg/g of Hb. These levels of CA-II were about 11% of those of 49-week-old female WL-chickens. Simple linear regression analysis showed significant associations between the level of CA-II and egg laying rate from 16 week-old at 63 week-old WL-chicken (p Conclusions Developmental changes and sexual differences of CA-II concentration in WL-chicken erythrocytes were observed. The concentration of CA-II in

  11. Research progress of carbon dioxide capture by using carbonic anhydrase%碳酸酐酶用于二氧化碳捕集的研究进展

    王静

    2012-01-01

    碳酸酐酶(CA)可以加速捕集化石燃料燃烧产生的二氧化碳,从而降低CO2的排放量.主要介绍了CA的来源、活性、稳定性及作用.分析了使用新型生物方法对二氧化碳进行捕集和储存的优缺点,并对下一步的工作进行了展望.%It has been demonstrated that carbonic anhydrase has the potential of accelerating of carbon dioxide capture from fossil fuel and reduce the discharge of carbon dioxide. The source, activity, stability and functions of carbonic anhydrase are mainly presented. In addition, the advantages and disadvantages of using new biological for carbon dioxide capture and storage are discussed and analyzed, and the further study is prospected.

  12. Carbonic anhydrase-related protein XI: structure of the gene in the greater false vampire bat (Megaderma lyra) compared with human and domestic pig.

    Porter, Calvin A; Hewett-Emmett, David; Tashian, Richard E

    2013-06-01

    Carbonic anhydrase-related protein XI (CA-RP XI) is a member of the α-carbonic anhydrase family (encoded by the gene CA-11), which has lost features of the active site required for enzymatic activity. Using PCR, we amplified CA-11 from genomic DNA of the bat Megaderma lyra. To elucidate the gene structure, we sequenced PCR products and compared their sequences with genomic and mRNA sequences known from human and domestic pig. We identified and sequenced eight introns in the bat CA-11. Five introns (introns 3-7) are located in identical or similar positions in other members of the vertebrate α-carbonic anhydrase gene family. Two 5' introns and one 3' intron are located in the regions of little or no sequence similarity with other members of the gene family. The low sequence similarity and additional introns suggest a separate evolutionary origin for the 5' and 3' portions of the CA-RP XI gene. PMID:23417223

  13. Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

    Werner E G Müller

    Full Text Available Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC. Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative enzyme carbonic anhydrase and the (putative osteoclast-stimulating factor (OSTF, that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2 show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+-depletion condition (1 mM CaCl(2. Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes to human (osteoclast.

  14. Preliminary X-ray crystallographic analysis of β-carbonic anhydrase psCA3 from Pseudomonas aeruginosa

    Two crystal forms of β-carbonic anhydrase psCA3 from P. aeruginosa were grown. Crystal form A belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and diffracted X-rays to 2.9 Å resolution; crystal form B belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and diffracted X-rays to 3.0 Å resolution. Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional β-carbonic anhydrases (β-CAs): psCA1, psCA2 and psCA3, respectively. These β-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of β-CA psCA3. Form A diffracted to a resolution of 2.9 Å; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and had a calculated Matthews coefficient of 2.23 Å3 Da−1 assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 Å; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and had a calculated Matthews coefficient of 2.48 Å3 Da−1 assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress

  15. Functional characterization of mutants affected in the carbonic anhydrase domain of the respiratory complex I in Arabidopsis thaliana.

    Soto, Débora; Córdoba, Juan Pablo; Villarreal, Fernando; Bartoli, Carlos; Schmitz, Jessica; Maurino, Veronica G; Braun, Hans Peter; Pagnussat, Gabriela C; Zabaleta, Eduardo

    2015-09-01

    The NADH-ubiquinone oxidoreductase complex (complex I) (EC 1.6.5.3) is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animals and fungi (Opisthokonta), it contains an extra domain comprising trimers of putative γ-carbonic anhydrases, named the CA domain, which has been proposed to be essential for assembly of complex I. However, its physiological role in plants is not fully understood. Here, we report that Arabidopsis mutants defective in two CA subunits show an altered photorespiratory phenotype. Mutants grown in ambient air show growth retardation compared to wild-type plants, a feature that is reversed by cultivating plants in a high-CO2 atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contributes to sustaining efficient photosynthesis under ambient (photorespiratory) conditions. PMID:26148112

  16. 植物碳酸酐酶的研究进展%Progress in Research on Plant Carbonic Anhydrase

    蒋春云; 马秀灵; 沈晓艳; 李燕; 赵彦修

    2013-01-01

    在植物组织中,碳酸酐酶(CA)催化CO2与HCO3-之间可逆的水合反应,重新固定呼吸释放的CO2并用于细胞光合作用.本文简要介绍了CA的生理机能、分类、亚细胞定位、基因功能等的研究进展,并展望了CA在提高C3植物光合效率以及CA在C3植物由C3光合类型转向C4光合类型方面的研究意义.%Carbonic anhydrase (CA) catalyses the reversible reaction between CO2 and HCO3-in plant living organisms.It can refix the respiration-released CO2 which participates in photosynthesis process.In this article we summarize the research progress in the physiological function,classification,subcellular localization and gene function of CA.And we prospect its crucial roles in increasing the photosynthetic rate in C3 plants and in the type of photosynthesis from C3 to C4.

  17. Synthesis of a new series of dithiocarbamates with effective human carbonic anhydrase inhibitory activity and antiglaucoma action.

    Bozdag, Murat; Carta, Fabrizio; Vullo, Daniela; Akdemir, Atilla; Isik, Semra; Lanzi, Cecilia; Scozzafava, Andrea; Masini, Emanuela; Supuran, Claudiu T

    2015-05-15

    A new series of dithiocarbamates (DTCs) was prepared from primary/secondary amines incorporating amino/hydroxyl-alkyl, mono- and bicyclic aliphatic ring systems based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, and carbon disulfide. The compounds were investigated for the inhibition of four mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, that is, the human (h) hCA I, II, IX and XII, drug targets for antiglaucoma (hCA II and XII) or antitumor (hCA IX/XII) agents. The compounds were moderate or inefficient hCA I inhibitors (off-target isoform for both applications), efficiently inhibited hCA II, whereas some of them were low nanomolar/subnanomolar hCA IX/XII inhibitors. One DTC showed excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, with a two times better efficiency compared to the clinically used sulfonamide dorzolamide. PMID:25846066

  18. Impacts of Elevated CO2 Concentration on Biochemical Composition,Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

    Jian-Rong XIA; Kun-Shan GAO

    2005-01-01

    To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations,Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2concentrations within the range 3-186 μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

  19. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms.

    Carradori, Simone; Secci, Daniela; De Monte, Celeste; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Codispoti, Rossella; De Cosmi, Federica; Guglielmi, Paolo; Supuran, Claudiu T

    2016-03-01

    Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with KIs ranging between 19 and 2482nM, whereas they were poorly active against hCA II (KIs >10μM) and hCA I (KIs ranging between 318nM and 50μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors. PMID:26810710

  20. Sulfonamide inhibition studies of the β-carbonic anhydrase from the newly discovered bacterium Enterobacter sp. B13.

    Eminoğlu, Ayşenur; Vullo, Daniela; Aşık, Aycan; Çolak, Dilşat Nigar; Çanakçı, Sabriye; Beldüz, Ali Osman; Supuran, Claudiu T

    2016-04-01

    The genome of the newly identified bacterium Enterobacter sp. B13 encodes for a β-class carbonic anhydrases (CAs, EC 4.2.1.1), EspCA. This enzyme was recently cloned, and characterized kinetically by this group (J. Enzyme Inhib. Med. Chem. 2016, 31). Here we report an inhibition study with sulfonamides and sulfamates of this enzyme. The best EspCA inhibitors were some sulfanylated sulfonamides with elongated molecules, metanilamide, 4-aminoalkyl-benzenesulfonamides, acetazolamide, and deacetylated methazolamide (KIs in the range of 58.7-96.5nM). Clinically used agents such as methazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, zonisamide, sulthiame, sulpiride, topiramate and valdecoxib were slightly less effective inhibitors (KIs in the range of 103-138nM). Saccharin, celecoxib, dichlorophenamide and many simple benzenesulfonamides were even less effective as EspCA inhibitors, with KIs in the range of 384-938nM. Identification of effective inhibitors of this bacterial enzyme may lead to pharmacological tools useful for understanding the physiological role(s) of the β-class CAs in bacterial pathogenicity/virulence. PMID:26920803

  1. Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies.

    Carta, Fabrizio; Ferraroni, Marta; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-15

    Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (KIs of 366-127 nM), CA IX (KIs of 8.1-36.9 nM), CA XII (KIs of 4.1-20.5 nM) and CA XIV (KIs of 12.8-53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies. PMID:26682703

  2. The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives.

    Taslimi, Parham; Gulcin, Ilhami; Ozgeris, Bunyamin; Goksu, Suleyman; Tumer, Ferhan; Alwasel, Saleh H; Supuran, Claudiu T

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66-4.14 nM) and hCA II inhibitors (Kis of 1.37-3.12 nM) and perfect AChE inhibitors (Kis in the range of 1.87-7.53 nM) compared to acetazolamide as CA inhibitor (Ki: 6.76 nM for hCA I and Ki: 5.85 nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64 nM). PMID:25697270

  3. Sulfonamide inhibition studies of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2, TcruCA.

    Vullo, Daniela; Bhatt, Avni; Mahon, Brian P; McKenna, Robert; Supuran, Claudiu T

    2016-01-15

    We report a sulfonamide/sulfamate inhibition study of the α-carbonic anhydrase (CA, EC 4.2.1.1) present in the gammaproteobacterium Thiomicrospira crunogena XCL-2, a mesophilic hydrothermal vent-isolate organism, TcruCA. As Thiomicrospira crunogena is one of thousands of marine organisms that uses CA for metabolic regulation, the effect of sulfonamide inhibition has been considered. Sulfonamide-based drugs have been widely used in a variety of antibiotics, and bioelimination of these compounds results in exposure of these compounds to marine life. The enzyme was highly inhibited, with Ki values ranging from 2.5 to 40.7nM by a variety of sulfonamides including acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide and benzenesulfonamides incorporating 4-hydroxyalkyl moieties. Less effective inhibitors were topiramate, zonisamide, celecoxib, saccharin and hydrochlorothiazide as well as simple benzenesulfonamides incorporating amino, halogeno, alkyl, aminoalkyl and other moieties in the ortho- or para-positions of the aromatic ring (Kis of 202-933nM). The active site interactions between TcruCA and three clinically-used CA inhibitors, acetazolamide (Diamox®), dorzolamide (Trusopt®), and brinzolamide (Azopt®) are studied using molecular docking to provide insight into the reported Ki values. Comparison between various enzymes belonging to this family may also bring interesting hints in these fascinating phenomena. PMID:26691758

  4. An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase Inhibition, and Protein X-ray Structures of Psammaplin C.

    Mujumdar, Prashant; Teruya, Kanae; Tonissen, Kathryn F; Vullo, Daniela; Supuran, Claudiu T; Peat, Thomas S; Poulsen, Sally-Ann

    2016-06-01

    Psammaplin C is one of only two described natural product primary sulfonamides. Here we report the synthesis of psammaplin C and evaluate the inhibition profile against therapeutically relevant carbonic anhydrase (CA) zinc metalloenzymes. The compound exhibited unprecedented inhibition of an important cancer-associated isozyme, hCA XII, with a Ki of 0.79 nM. The compound also displayed good isoform selectivity for hCA XII over other CAs. We present the first reported protein X-ray crystal structures of psammaplin C in complex with human CAs. We engineered the easily crystallized hCA II enzyme to mimic both the hCA IX and hCA XII binding sites and then utilized protein X-ray crystallography to determine the binding pose of psammaplin C within the hCA II, hCA IX, and hCA XII mimic active sites, all to high resolution. This is the first time a natural product primary sulfonamide inhibitor has been assessed for inhibition and binding to CAs. PMID:27172398

  5. Effects of novel auto-inducible medium on growth, activity and CO₂ capture capacity of Escherichia coli expressing carbonic anhydrase.

    Watson, Stuart K; Kan, Eunsung

    2015-10-01

    A glucose-based auto-inducible medium (glucose-AIM) has been developed to enhance both growth and expression of lac operon-linked carbonic anhydrase (CA) expression in a recombinant strain of Escherichia coli. When the E. coli expressing CA was grown on various media, the glucose-based auto-inducible medium (glucose AIM) resulted in a CA activity of 1022 mU OD(600 nm)(-1) mL(-1) at 24 h and a specific growth rate of 0.082 h(-1). The CA activity was four to fourteen times higher than those by LB-IPTG. The E. coli expressing CA grown on the glucose-AIM showed highest activity at pH8.5 while it kept high stability up to 40°C and an inlet CO2 concentration of 6%. These findings indicate that the glucose-AIM would be a cost-effective medium to support high cell growth, CA activity and stability for effective CO2 capture. PMID:26264623

  6. A Divalent PAMAM-Based Matrix Metalloproteinase/Carbonic Anhydrase Inhibitor for the Treatment of Dry Eye Syndrome.

    Richichi, B; Baldoneschi, V; Burgalassi, S; Fragai, M; Vullo, D; Akdemir, A; Dragoni, E; Louka, A; Mamusa, M; Monti, D; Berti, D; Novellino, E; De Rosa, G; Supuran, C T; Nativi, C

    2016-01-26

    Synthetic sulfonamide derivatives are a class of potent matrix metalloproteinase inhibitors (MMPI) that have potential for the treatment of diseases related to uncontrolled expression of these enzymes. The lack of selectivity of the large majority of such inhibitors, leading to the inhibition of MMPs in tissues other than the targeted one, has dramatically reduced the therapeutic interest in MMPIs. The recent development of efficient drug delivery systems that allow the transportation of a selected drug to its site of action has opened the way to new perspectives in the use of MMPIs. Here, a PAMAM-based divalent dendron with two sulfonamidic residues was synthesized. This nanomolar inhibitor binds to the catalytic domain of two MMPs as well as to the transmembrane human carbonic anhydrases (hCAs) XII, which is present in the eye and considered an antiglaucoma target. In the animal model of an experimental dry eye, no occurrence of dotted staining in eyes treated with our inhibitor was observed, indicating no symptoms of corneal desiccation. PMID:26692423

  7. Evaluation of a Carbonic Anhydrase IX-Targeted Near-Infrared Dye for Fluorescence-Guided Surgery of Hypoxic Tumors.

    Lv, Peng-Cheng; Roy, Jyoti; Putt, Karson S; Low, Philip S

    2016-05-01

    Proof-of-principle studies in ovarian, lung, and brain cancer patients have shown that fluorescence-guided surgery can enable removal of otherwise undetectable malignant lesions, decrease the number of cancer-positive margins, and permit identification of disease-containing lymph nodes that would have normally evaded resection. Unfortunately, the current arsenal of tumor-targeted fluorescent dyes does not permit identification of all cancers, raising the need to design new tumor-specific fluorescent dyes to illuminate the currently undetectable cancers. In an effort to design a more universal fluorescent cancer imaging agent, we have undertaken to synthesize a fluorophore that could label all hypoxic regions of tumors. We report here the synthesis, in vitro binding, and in vivo imaging of a near-infrared (NIR) fluorescent dye that is targeted to carbonic anhydrase IX (CA IX), i.e., a widely accepted marker of hypoxic tissues. The low molecular weight NIR probe, named Hypoxyfluor, is shown to bind CA IX with high affinity and accumulate rapidly and selectively in CA IX positive tumors. Because nearly all human cancers contain hypoxic regions that express CA IX abundantly, this NIR probe should facilitate surgical resection of a wide variety of solid tumors. PMID:27043317

  8. Role of Carbonic Anhydrase as an Activator in Carbonate Rock Dissolution and Its Implication for Atmospheric CO2 Sink

    刘再华

    2001-01-01

    The conversion of CO2 into H+ and is a relatively slow reaction. Hence, its kinetics may be rate determining in carbonate rock dissolution. Carbonic anhydrase (CA), which is widespread in nature, was used to catalyze the CO2 conversion process in dissolution experiments of limestone and dolomite. It was found that the rate of dissolution increases by a factor of about 10 after the addition of CA at a high CO2 partial pressure (Pco2) for limestone and about 3 at low Pco2 for dolomite. This shows that reappraisal is necessary for the importance of chemical weathering (including carbonate rock dissolution and silicate weathering) in the atmospheric CO2 sink and the mysterious missing sink in carbon cycling. It is doubtless that previous studies of weathering underestimated weathering rates due to the ignorance of CA as an activator in weathering, thus the contribution of weathering to the atmospheric CO2 sink is also underestimated. This finding also shows the need to examine the situ distribution and activity of CA in different waters and to investigate the role of CA in weathering.``

  9. Carbonic Anhydrase VI Gene Polymorphism rs2274327 Relationship Between Salivary Parameters and Dental-Oral Health Status in Children.

    Sengul, Fatih; Kilic, Munevver; Gurbuz, Taskin; Tasdemir, Sener

    2016-08-01

    The aim of this study was to research carbonic anhydrase (CA) VI one single-nucleotide polymorphism (SNP) and its potential association with dental-oral health status (dental caries, Plaque Index (PI) and Gingival Index (GI)) and salivary parameters (salivary buffering capacity, salivary flow rate (SFR)) in children. A total of 178 children were divided into two groups: non-carious (n = 70, 34 boys and 36 girls) and carious (n = 108, 47 boys and 61 girls). The clinical evaluations were performed according to the decayed, missing, and filled teeth (dmft/DMFT) index by a specialist. Clinical parameters including PI, GI, and simplified oral hygiene index (OHI-S) were recorded. Salivary pH (SpH) was measured using pH paper. Blood samples and unstimulated whole saliva were collected, and SFR was calculated. The CA VI rs2274327 polymorphism was determined by a LightSNiP assay on the realtime PCR system. The frequencies of rs2274327 were not significant between groups (p > 0.05). There was a positive correlation between OHI-S and SpH in the carious and non-carious groups (p  0.05). CA VI SNP (rs2274327) had no statistically significant association with OHI-S, PI, GI, SFR, and SpH in the children. PMID:27100223

  10. Effects of intraleaf variations in carbonic anhydrase activity and gas exchange on leaf C18OO isoflux in Zea mays.

    Affek, Hagit P; Krisch, Maria J; Yakir, Dan

    2006-01-01

    Variation in the C18OO content of atmospheric CO2 (delta18Oa) can be used to distinguish photosynthesis from soil respiration, which is based on carbonic anhydrase (CA)-catalyzed 18O exchange between CO2 and 18O-enriched leaf water (delta18Ow). Here we tested the hypothesis that mean leaf delta18Ow and assimilation rates can be used to estimate whole-leaf C18OO flux (isoflux), ignoring intraleaf variations in CA activity and gas exchange parameters. We observed variations in CA activity along the leaf (> 30% decline from the leaf center toward the leaf ends), which were only partially correlated to those in delta18Ow (7 to 21 per thousand), delta18O and delta13C of leaf organic matter (25 to 30 per thousand and -12.8 to -13.2 per thousand, respectively), and substomatal CO2 concentrations (intercellular CO2 concentrations, c(i), at the leaf center were approximately 40% of those at the leaf tip). The combined effect of these variations produced a leaf-integrated isoflux that was different from that predicted based on bulk leaf values. However, because of canceling effects among the influencing parameters, isoflux overestimations were only approximately 10%. Conversely, use of measured parameters from a leaf segment could produce large errors in predicting leaf-integrated C18OO fluxes. PMID:16411935