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Sample records for carbonic anhydrase cah3

  1. Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii.

    PubMed

    Benlloch, Reyes; Shevela, Dmitriy; Hainzl, Tobias; Grundström, Christin; Shutova, Tatyana; Messinger, Johannes; Samuelsson, Göran; Sauer-Eriksson, A Elisabeth

    2015-03-01

    In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 (-) from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 (-) on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration. PMID:25617045

  2. Natural products that inhibit carbonic anhydrase.

    PubMed

    Poulsen, Sally-Ann; Davis, Rohan A

    2014-01-01

    The chemical diversity, binding specificity and propensity to interact with biological targets has inspired many researchers to utilize natural products as molecular probes. Almost all reported carbonic anhydrase inhibitors comprise a zinc binding group in their structure of which the primary sulfonamide moiety (-SO2NH2) is the foremost example and to a lesser extent the primary sulfamate (-O-SO2NH2) and sulfamide (-NH-SO2NH2) groups. Natural products that comprise these zinc binding groups in their structure are however rare and relatively few natural products have been explored as a source for novel carbonic anhydrase inhibitors. This chapter will highlight the recent and growing interest in carbonic anhydrase inhibitors sourced from nature, demonstrating that natural product chemical space presents a rich source of potential alternate chemotypes for the discovery of novel drug-like carbonic anhydrase inhibitors. PMID:24146386

  3. Accelerating Mineral Carbonation Using Carbonic Anhydrase.

    PubMed

    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.

  4. Accelerating Mineral Carbonation Using Carbonic Anhydrase.

    PubMed

    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

  5. Prokaryotic carbonic anhydrases of Earth's environment.

    PubMed

    Kumar, R Siva Sai; Ferry, James G

    2014-01-01

    Carbonic anhydrase is a metalloenzyme catalyzing the reversible hydration of carbon dioxide to bicarbonate. Five independently evolved classes have been described for which one or more are found in nearly every cell type underscoring the general importance of this ubiquitous enzyme in Nature. The bulk of research to date has centered on the enzymes from mammals and plants with less emphasis on prokaryotes. Prokaryotic carbonic anhydrases play important roles in the ecology of Earth's biosphere including acquisition of CO2 for photosynthesis and the physiology of aerobic and anaerobic prokaryotes decomposing the photosynthate back to CO2 thereby closing the global carbon cycle. This review focuses on the physiology and biochemistry of carbonic anhydrases from prokaryotes belonging to the domains Bacteria and Archaea that play key roles in the ecology of Earth's biosphere.

  6. Carbonic anhydrase inhibitors drug design.

    PubMed

    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

  7. Thermostable Carbonic Anhydrases in Biotechnological Applications

    PubMed Central

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

    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, 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. PMID:26184158

  8. Chemically modified carbonic anhydrases useful in carbon capture systems

    SciTech Connect

    Novick, Scott; Alvizo, Oscar

    2013-01-15

    The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

  9. The effect of bile salts on carbonic anhydrase.

    PubMed

    Milov, D E; Jou, W S; Shireman, R B; Chun, P W

    1992-02-01

    Bile salts are potent inhibitors of bovine carbonic anhydrase and human carbonic anhydrase I and human carbonic anhydrase II. To further characterize the binding of bile salts to carbonic anhydrase, rate constants for the CO2 hydration reaction in the presence of deoxycholate, cholate, glycocholate and taurocholate were determined using stop-flow experiments. Values for the Michaelis-Menton dissociation constant for bovine carbonic anhydrase, human carbonic anhydrase I and human carbonic anhydrase II were found to be 5.2, 9.2 and 13.2 mmol/L, respectively. The inhibition constant values for the various bile salts tested ranged from 0.1 to 1 mmol/L for bovine carbonic anhydrase, 1.6 to 2.4 mmol/L for human carbonic anhydrase I and 0.09 to 0.7 mmol/L for human carbonic anhydrase II. Our results suggest a mechanism of noncompetitive carbonic anhydrase inhibition for bile salts. Bile-salt binding to carbonic anhydrases as measured by scanning molecular sieve chromatography resulted in an increase in partition radius, molecular volume and surface area. The partition radius increased from 24 A to 28 A in the presence of 2.5 mmol/L sodium deoxycholate at critical micelle concentration. As determined by sedimentation equilibrium measurements, approximately 1 gm of carbonic anhydrase will bind 0.03 gm of deoxycholate, suggesting three to six binding sites for bile salt on the carbonic anhydrase molecule. The conformational changes and inhibition of carbonic anhydrases resulting from bile-salt binding may be important to the regulation of enzymatic activity in tissues along the enterohepatic circulation; by limiting bicarbonate availability this interaction may also contribute to the metabolic derangements seen in patients with cholestatic liver disease. PMID:1735532

  10. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    PubMed

    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

  11. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    PubMed

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

    2016-06-03

    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.

  12. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

    PubMed Central

    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

  13. Physiological functions of the alpha class of carbonic anhydrases.

    PubMed

    Frost, Susan C

    2014-01-01

    Carbonic anhydrases are ubiquitous enzymes that catalyze the reversible hydration of carbon dioxide. These enzymes are of ancient origin as they are found in the deepest of branches of the evolutionary tree. Of the five different classes of carbonic anhydrases, the alpha class has perhaps received the most attention because of its role in human pathology. This review focuses on the physiological function of this class of carbonic anhydrases organized by their cellular location.

  14. Non-Classical Inhibition of Carbonic Anhydrase

    PubMed Central

    Lomelino, Carrie L.; Supuran, Claudiu T.; McKenna, Robert

    2016-01-01

    Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives. PMID:27438828

  15. Carborane-based carbonic anhydrase inhibitors.

    PubMed

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

    2013-12-16

    CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors. PMID:24307504

  16. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques specific carbonic anhydrase protein molecules in serum and other body fluids. Measurements of carbonic anhydrase B and C aid in the diagnosis of abnormal hemoglobin metabolism....

  17. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques specific carbonic anhydrase protein molecules in serum and other body fluids. Measurements of carbonic anhydrase B and C aid in the diagnosis of abnormal hemoglobin metabolism....

  18. Radioimmunoassay of carbonic anhydrase III in rat tissues.

    PubMed Central

    Shiels, A; Jeffery, S; Wilson, C; Carter, N

    1984-01-01

    A specific and sensitive radioimmunoassay for the rat carbonic anhydrase III isoenzyme was developed. High concentrations of carbonic anhydrase III were detected in soleus muscle and male liver. Female liver and other skeletal muscles contained significantly lower concentrations, and only trace amounts were found in heart, prostate, kidney, brain, plasma, urine and, possibly, erythrocytes. PMID:6424658

  19. The Catalytic Mechanism of Carbonic Anhydrase

    PubMed Central

    Lindskog, Sven; Coleman, Joseph E.

    1973-01-01

    It is shown that an “inverse” relationship between the pH dependencies of the rates of hydration of CO2 and dehydration of HCO3- by carbonic anhydrase (EC 4.2.1.1) is a direct consequence of the thermodynamic equilibrium between CO2 and HCO3- and independent of any assumptions about the catalytic mechanism. It is further shown that proposed mechanisms for carbonic anhydrase involving HCO3- as the substrate in the dehydration reaction and a proton transfer reaction, EH+ ⇌ E + H+, as an obligatory step during catalysis obey the rule of microscopic reversibility. This includes mechanisms in which the proton dissociation is from a zinc-coordinated water molecule. Such mechanisms can be in accord with the observed rapid turnover rates of the enzyme, since rapid proton exchange can occur with the buffer components, EH+ + B ⇌ E + BH+. Mechanisms in which H2CO3 is the substrate in dehydration avoid the proton-transfer step, but require that H2CO3 combines with enzyme more rapidly than in a diffusion-controlled reaction. Physico-chemical evidence for and against a zinc-hydroxide mechanism is discussed. PMID:4200327

  20. Carbonic Anhydrases and Their Biotechnological Applications

    PubMed Central

    Boone, Christopher D.; Habibzadegan, Andrew; Gill, Sonika; McKenna, Robert

    2013-01-01

    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. PMID:24970180

  1. Carbonic Anhydrase Catalysis: An Experiment on Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Spyridis, Greg T.; And Others

    1985-01-01

    Describes an undergraduate enzyme kinetics experiment which uses bovine erythrocyte carbonic anhydrase, a very stable enzyme commercially available in lyophilized form. Includes background information, reactions involved, procedures used, and the calculation of typical results obtained. (JN)

  2. Carbonic Anhydrase and Metalloderivatives: A Bioinorganic Chemistry Study

    ERIC Educational Resources Information Center

    McQuate, Robert S.

    1977-01-01

    Discusses selected bioinorganic aspects of carbonic anhydrase and describes experiments that will reinforce the students' understanding of the presence and essential role that metal ions have in some biological systems. (SL)

  3. A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains.

    PubMed

    Leggat, William; Dixon, Ross; Saleh, Said; Yellowlees, David

    2005-07-01

    This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.

  4. Structure and function of carbonic anhydrases.

    PubMed

    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

  5. Azosulfonamides: Preparation and Binding to Carbonic Anhydrase: A Bioorganic Chemistry Experiment.

    ERIC Educational Resources Information Center

    Manalang, Mary G.; Bundy, Hallie F.

    1989-01-01

    Reports an experiment for examining protein-ligand binding using the enzyme carbonic anhydrase. Describes the experimental procedures consisting of the syntheses of an azosulfonamide and its complex formation with carbonic anhydrase. (YP)

  6. Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle.

    PubMed Central

    Bruns, W; Dermietzel, R; Gros, G

    1986-01-01

    Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0.1-0.4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.) Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-1). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 X 10(-8) M and similar but not identical to that of cytosolic carbonic anhydrase II. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates (Siffert & Gros, 1982) originates from the s.r. Histochemical studies using the dansylsuphonamide method described previously (Dermietzel, Leibstein, Siffert, Zamboglou & Gros, 1985) showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre cross-sections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres. It is estimated that s.r. carbonic anhydrase accelerates CO2 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3- react fast enough to provide a rapid source and sink for protons leaving

  7. How many carbonic anhydrase inhibition mechanisms exist?

    PubMed

    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

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

    SciTech Connect

    Fisher, S. Z. Kovalevsky, A. Y.; Domsic, J.; Mustyakimov, M.; Silverman, D. N.; McKenna, R.; Langan, P.

    2010-11-01

    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 CO{sub 2} to form HCO{sub 3}{sup −} and H{sup +} using a Zn–hydroxide mechanism. The first part of catalysis involves CO{sub 2} 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.

  9. Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

    ERIC Educational Resources Information Center

    Wang, Sheila C.; Zamble, Deborah B.

    2006-01-01

    A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

  10. Revisiting zinc coordination in human carbonic anhydrase II.

    PubMed

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

    2012-10-15

    Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA, CA where Zn(2+) ion has been removed) is relatively easy to generate, and 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 (K(a) ≈ 1.2 × 10(12)) for Zn(2+). However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn(2+) with a strong equilibrium constant (K(a) ≈ 2 × 10(9)) that is 3 orders of magnitude lower than the previously established value. Thermodynamic parameters associated with Zn(2+) binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and the reconstituted ZnCA (active CA containing a mononuclear Zn(2+) center) were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirm the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II. PMID:23030313

  11. Carbonic anhydrase-I polymorphism in a Philippine aboriginal population.

    PubMed Central

    Omoto, K

    1979-01-01

    Polymorphism of carbonic anhydrase-I (CA1) was found by electrophoresis in an aboriginal group of Mindanao, Philippines, with a remarkably high frequency of variant types. The frequency of the variant allele was estimated at .256. The variant isozyme designated CA1 3Negrito (CA1 3N) is electrophoretically indistinguishable from the "Guam" variant and may be regarded as a potential anthropological marker in the Western Pacific. Images Fig. 1 PMID:117701

  12. CO2 Removal using a Synthetic Analogue of Carbonic Anhydrase

    SciTech Connect

    Cordatos, Harry

    2010-09-14

    Project attempts to develop a synthetic analogue for carbonic anhydrase and incorporate it in a membrane for separation of CO2 from coal power plant flue gas. Conference poster presents result of first 9 months of project progress including concept, basic system architecture and membrane properties target, results of molecular modeling for analogue - CO2 interaction, and next steps of testing analogue resistance to flue gas contaminants.

  13. An Update on Natural Products with Carbonic Anhydrase Inhibitory Activity.

    PubMed

    Karioti, Anastasia; Carta, Fabrizio; Supuran, Claudiu T

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological processes. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. It is more than 70 years that synthetic compounds, mainly sulfonamides, have been used in clinical practice as diuretics and systemic acting antiglaucoma drugs. Recent studies using natural product libraries and isolated constituents from natural sources (such as fungi and plants) have disclosed novel chemotypes possessing carbonic anhydrase inhibition activities. These natural sources offer new opportunities in the search for new and more effective carbonic anhydrase inhibitors, and may serve as new leads for the design and development of future drugs. This review will discuss the most recent advances in the search of naturally occurring products and their synthetic derivatives that inhibit the CAs and their mechanisms of action at molecular level. Plant extracts are not considered in the present review. PMID:26654592

  14. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques specific carbonic anhydrase protein molecules in serum and other body fluids...) Classification. Class I (general controls). The device is exempt from the premarket notification procedures...

  15. Removal of Zinc Form Carbonic Anhydrase: A Kinetics Experiment for Upper-Level Chemistry Laboratories

    ERIC Educational Resources Information Center

    Williams, Kathryn R.; Adhyaru, Bhavin

    2004-01-01

    An experiment on kinetics of deactivation of carbonic anhydrase by removal of zinc is demonstrated. Carbonic anhydrase, the enzyme that catalyzes the interconversion of carbon dioxide and bicarbonate, requires on Zn(II) ion in its active site, and removal of the zinc cofactor by complexion to another ligand leaves the apoenzyme, which is totally…

  16. Carbon dioxide "trapped" in a β-carbonic anhydrase

    DOE PAGES

    Aggarwal, Mayank; Chua, Teck Khiang; Pinard, Melissa A.; Szebenyi, Doletha M.; McKenna, Robert

    2015-10-12

    Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/ dehydration of CO2/HCO3- with rates approaching diffusion-controlled limits (kcat/KM ~ 108 M–1s–1). Here, this family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a beta-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryocooled crystallography. The structure has been refined to 1.6 angstrom resolution with Rcryst and Rfree values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to havemore » CO2, captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2, binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2, binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dirners or higher-order oligomeric states, and the CO2, bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.« less

  17. Highly stable beta-class carbonic anhydrases useful in carbon capture systems

    DOEpatents

    Alvizo, Oscar; Benoit, Mike; Novick, Scott

    2013-04-16

    The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

  18. Highly stable beta-class carbonic anhydrases useful in carbon capture systems

    SciTech Connect

    Alvizo, Oscar; Benoit, Michael R; Novick, Scott J

    2013-08-20

    The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

  19. Bioinformatic analysis of beta carbonic anhydrase sequences from protozoans and metazoans

    PubMed Central

    2014-01-01

    Background Despite the high prevalence of parasitic infections, and their impact on global health and economy, the number of drugs available to treat them is extremely limited. As a result, the potential consequences of large-scale resistance to any existing drugs are a major concern. A number of recent investigations have focused on the effects of potential chemical inhibitors on bacterial and fungal carbonic anhydrases. Among the five classes of carbonic anhydrases (alpha, beta, gamma, delta and zeta), beta carbonic anhydrases have been reported in most species of bacteria, yeasts, algae, plants, and particular invertebrates (nematodes and insects). To date, there has been a lack of knowledge on the expression and molecular structure of beta carbonic anhydrases in metazoan (nematodes and arthropods) and protozoan species. Methods Here, the identification of novel beta carbonic anhydrases was based on the presence of the highly-conserved amino acid sequence patterns of the active site. A phylogenetic tree was constructed based on codon-aligned DNA sequences. Subcellular localization prediction for each identified invertebrate beta carbonic anhydrase was performed using the TargetP webserver. Results We verified a total of 75 beta carbonic anhydrase sequences in metazoan and protozoan species by proteome-wide searches and multiple sequence alignment. Of these, 52 were novel, and contained highly conserved amino acid residues, which are inferred to form the active site in beta carbonic anhydrases. Mitochondrial targeting peptide analysis revealed that 31 enzymes are predicted with mitochondrial localization; one was predicted to be a secretory enzyme, and the other 43 were predicted to have other undefined cellular localizations. Conclusions These investigations identified 75 beta carbonic anhydrases in metazoan and protozoan species, and among them there were 52 novel sequences that were not previously annotated as beta carbonic anhydrases. Our results will not

  20. Carbon dioxide "trapped" in a β-carbonic anhydrase

    SciTech Connect

    Aggarwal, Mayank; Chua, Teck Khiang; Pinard, Melissa A.; Szebenyi, Doletha M.; McKenna, Robert

    2015-10-12

    Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/ dehydration of CO2/HCO3- with rates approaching diffusion-controlled limits (kcat/KM ~ 108 M–1s–1). Here, this family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a beta-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryocooled crystallography. The structure has been refined to 1.6 angstrom resolution with Rcryst and Rfree values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2, captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2, binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2, binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dirners or higher-order oligomeric states, and the CO2, bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.

  1. Carbonic anhydrase inhibitors. Inhibition of the Rv1284 and Rv3273 beta-carbonic anhydrases from Mycobacterium tuberculosis with diazenylbenzenesulfonamides.

    PubMed

    Maresca, Alfonso; Carta, Fabrizio; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2009-09-01

    A series of diazenylbenzenesulfonamides obtained from sulfanilamide or metanilamide by diazotization followed by coupling with phenols or amines, was tested for the inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) encoded by the genes Rv1284 and Rv3273 of Mycobacterium tuberculosis. Several low micromolar inhibitors of the two enzymes were detected, with prontosil being the best inhibitor (K(I)s of 126-148nM). Inhibition of pathogenic beta-CAs may lead to the development of antiinfectives with a new mechanism of action, devoid of resistance problems encountered with classical antibiotics. PMID:19651511

  2. Building reactive copper centers in human carbonic anhydrase II.

    PubMed

    Song, He; Weitz, Andrew C; Hendrich, Michael P; Lewis, Edwin A; Emerson, Joseph P

    2013-08-01

    Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu(2+) ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu(2+)-binding events have different affinities (K a approximately 5 × 10(12) and 1 × 10(10)), and both are enthalpically driven processes. Reconstituting these Cu(2+) sites under a range of conditions has allowed us to assign the Cu(2+)-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu(2+) sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol. PMID:23744511

  3. A cytosolic carbonic anhydrase molecular switch occurs in the gills of metamorphic sea lamprey

    PubMed Central

    Ferreira-Martins, D.; McCormick, S. D.; Campos, A.; Lopes-Marques, M.; Osório, H.; Coimbra, J.; Castro, L. F. C.; Wilson, J. M.

    2016-01-01

    Carbonic anhydrase plays a key role in CO2 transport, acid-base and ion regulation and metabolic processes in vertebrates. While several carbonic anhydrase isoforms have been identified in numerous vertebrate species, basal lineages such as the cyclostomes have remained largely unexamined. Here we investigate the repertoire of cytoplasmic carbonic anhydrases in the sea lamprey (Petromyzon marinus), that has a complex life history marked by a dramatic metamorphosis from a benthic filter-feeding ammocoete larvae into a parasitic juvenile which migrates from freshwater to seawater. We have identified a novel carbonic anhydrase gene (ca19) beyond the single carbonic anhydrase gene (ca18) that was known previously. Phylogenetic analysis and synteny studies suggest that both carbonic anhydrase genes form one or two independent gene lineages and are most likely duplicates retained uniquely in cyclostomes. Quantitative PCR of ca19 and ca18 and protein expression in gill across metamorphosis show that the ca19 levels are highest in ammocoetes and decrease during metamorphosis while ca18 shows the opposite pattern with the highest levels in post-metamorphic juveniles. We propose that a unique molecular switch occurs during lamprey metamorphosis resulting in distinct gill carbonic anhydrases reflecting the contrasting life modes and habitats of these life-history stages. PMID:27703170

  4. Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro

    NASA Technical Reports Server (NTRS)

    Hall, G. E.; Kenny, A. D.

    1985-01-01

    The possible role of carbonic anhydrase in bone resorption induced by prostaglandin E2 (PGE2) was studied using an in vitro neonatal mouse calvarial culture system. PGE2 (10 to the -6th M) was effective in stimulating resorption, as assessed by calcium release into culture media. This enhanced resorption was accompanied by significant increases in calvarial carbonic anhydrase activity over control values at 48 and 96 h. At 48 h, bones treated with PGE2 had 20 percent more carbonic anhydrase activity than controls. By 96 h, treated bones contained 79 percent more carbonic anhydrase activity than controls. PGE2-induced bone resorption was inhibited by the carbonic anhydrase inhibitor acetazolamide in a dose-dependent fashion from 10 to the -5th to 10 to the -4th M with 77 percent inhibition observed at 10 to the -4th M. The acetazolamide analogue CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase, failed to inhibit PGE2-induced resorption. These results are consistent with the hypothesis that carbonic anhydrase is a necessary component of the osteoclastic bone resorptive mechanism.

  5. Evolution of carbonic anhydrase in C4 plants.

    PubMed

    Ludwig, Martha

    2016-06-01

    During the evolution of C4 photosynthesis, the intracellular location with most carbonic anhydrase (CA) activity has changed. In Flaveria, the loss of the sequence encoding a chloroplast transit peptide from an ancestral C3 CA ortholog confined the C4 isoform to the mesophyll cell cytosol. Recent studies indicate that sequence elements and histone modifications controlling the expression of C4-associated CAs were likely present in the C3 ancestral chromatin, enabling the evolution of the C4 pathway. Almost complete abolishment of maize CA activity yields no obvious phenotype at ambient CO2 levels. This contrasts with results for Flaveria CA mutants, and has opened discussion on the role of CA in the C4 carbon concentrating mechanism.

  6. Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum.

    PubMed

    Dixon, G K; Patel, B N; Merrett, M J

    1987-12-01

    Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO2-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O2 evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO 3 (-) . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O2 evolution (K0.5[CO2]). At pH 8.0, where HCO 3 (-) is the predominant species of inorganic carbon, the K0.5 (CO2) was increased from 50 μM to 950 μM in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO 3 (-) is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO2 from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO 3 (-) and CO2 within the cell.

  7. The role of carbonic anhydrase in the pathogenesis of vascular calcification in humans.

    PubMed

    Adeva-Andany, María M; Fernández-Fernández, Carlos; Sánchez-Bello, Rocío; Donapetry-García, Cristóbal; Martínez-Rodríguez, Julia

    2015-07-01

    Carbonic anhydrases are a group of isoenzymes that catalyze the reversible conversion of carbon dioxide into bicarbonate. They participate in a constellation of physiological processes in humans, including respiration, bone metabolism, and the formation of body fluids, including urine, bile, pancreatic juice, gastric secretion, saliva, aqueous humor, cerebrospinal fluid, and sweat. In addition, carbonic anhydrase may provide carbon dioxide/bicarbonate to carboxylation reactions that incorporate carbon dioxide to substrates. Several isoforms of carbonic anhydrase have been identified in humans, but their precise physiological role and the consequences of their dysfunction are mostly unknown. Carbonic anhydrase isoenzymes are involved in calcification processes in a number of biological systems, including the formation of calcareous spicules from sponges, the formation of shell in some animals, and the precipitation of calcium salts induced by several microorganisms, particularly urease-producing bacteria. In human tissues, carbonic anhydrase is implicated in calcification processes either directly by facilitating calcium carbonate deposition which in turn serves to facilitate calcium phosphate mineralization, or indirectly via its action upon γ-glutamyl-carboxylase, a carboxylase that enables the biological activation of proteins involved in calcification, such as matrix Gla protein, bone Gla protein, and Gla-rich protein. Carbonic anhydrase is implicated in calcification of human tissues, including bone and soft-tissue calcification in rheumatological disorders such as ankylosing spondylitis and dermatomyositis. Carbonic anhydrase may be also involved in bile and kidney stone formation and carcinoma-associated microcalcifications. The aim of this review is to evaluate the possible association between carbonic anhydrase isoenzymes and vascular calcification in humans. PMID:26005791

  8. Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

    PubMed

    Guilloton, M B; Korte, J J; Lamblin, A F; Fuchs, J A; Anderson, P M

    1992-02-25

    The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.

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

    PubMed

    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

  10. Characterization of human carbonic anhydrase III from skeletal muscle.

    PubMed

    Carter, N; Jeffery, S; Shiels, A; Edwards, Y; Tipler, T; Hopkinson, D A

    1979-10-01

    A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.

  11. Kinetic and structural characterization of spinach carbonic anhydrase.

    PubMed

    Rowlett, R S; Chance, M R; Wirt, M D; Sidelinger, D E; Royal, J R; Woodroffe, M; Wang, Y F; Saha, R P; Lam, M G

    1994-11-29

    We have carried out kinetics studies of spinach carbonic anhydrase (CA) using stopped-flow spectrophotometry at steady state and 13C-NMR exchange at chemical equilibrium. We found that the rate of CO2<-->HCO3- exchange catalyzed by spinach CA at pH 7.0 to be 3-5 times faster than the maximal kcat for either CO2 hydration or HCO3- dehydration at steady state, suggesting a rate-determining H+ transfer step in the catalytic mechanism. Correspondingly, we measured a pH-independent solvent deuterium isotope effect on kcat of approximately 2.0, and found that the rate of catalysis was significantly decreased at external buffer concentrations below 5 mM. Our results are consistent with a zinc-hydroxide mechanism of action with for spinach CA, similar to that of animal carbonic anhydrases. We have also collected X-ray absorption spectra of spinach CA. Analysis of the extended fine structure (EXAFS) suggests that the coordination sphere of Zn in spinach CA must have one or more sulfur ligands, in contrast to animal CAs which have only nitrogen and oxygen ligands. The models which best fit the data have average Zn-N(O) distances of 1.99-2.06 A, average Zn-S distances of 2.31--2.32 A, and a total coordination number of 4-6. We conclude that animal and spinach CAs are convergently evolved enzymes which are structurally quite different, but functionally equivalent. PMID:7947805

  12. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    SciTech Connect

    Katzman, G.; Togasaki, R.K. ); Marcus, Y. ); Moroney, J.V. )

    1989-04-01

    In a new assay of carbonic anhydrase, NaH{sup 14}CO{sub 3} solution at the bottom of a sealed vessel releases {sup 14}CO{sub 3} 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 {plus minus} 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 {plus minus} 1.9 and 2.8 {plus minus} 7.8 WAU/mg chl respectively. Chloroplasts isolated from CWD and CIA-3/CW15 and subsequently disrupted gave 64.0 {plus minus} 14.7 and 2.8 {plus minus} 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.

  13. Sulfamates of methyl triterpenoates are effective and competitive inhibitors of carbonic anhydrase II.

    PubMed

    Schwarz, Stefan; Sommerwerk, Sven; Lucas, Susana D; Heller, Lucie; Csuk, René

    2014-10-30

    Carbonic anhydrase II, belonging to one of the most important enzyme groups of the human body, is a well-studied isozyme from the family of the carbonic anhydrases. Since it is involved in several physiological processes, it has been a pharmaceutical target for many years. In this study we synthesized a number of sulfamates derived from pentacyclic methyl triterpenoates, and we demonstrate their potential as carbonic anhydrase II inhibitors using the well-established photometric 4-nitrophenyl acetate assay. Inhibition constants, as an indicator of their inhibition strength, were in the micromolar range; one compound (10, methyl (3β) 3-(aminosulfonyloxy)-oleanoate) showed a Ki value as low as 0.3 μM. This Ki value is comparable to that of acetazolamide which is a potent carbonic anhydrase inhibitor and a drug for the treatment of glaucoma.

  14. Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity.

    PubMed

    Uda, Narasimha Rao; Seibert, Volker; Stenner-Liewen, Frank; Müller, Philipp; Herzig, Petra; Gondi, Gabor; Zeidler, Reinhard; van Dijk, Marc; Zippelius, Alfred; Renner, Christoph

    2015-12-01

    Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase's. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases. PMID:25775095

  15. Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

    PubMed

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

    2014-09-15

    The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report. PMID:25168745

  16. Carbonic anhydrase inhibitors: inhibition of cytosolic carbonic anhydrase isozymes II and VII with simple aromatic sulfonamides and some azo dyes.

    PubMed

    Carta, Fabrizio; Pothen, Blessy; Maresca, Alfonso; Tiwari, Meena; Singh, Vineet; Supuran, Claudiu T

    2009-08-01

    Several substituted benzenesulfonamides were synthesized by various pathways starting from sulfanilamide. The sulfanilamide diazonium salt was reacted with copper (I) halides, potassium iodide and/or aromatic derivatives, leading to 4-halogeno-, and 4-hydroxy-benzenesulfonamides as well as diazo dyes incorporating sulfamoyl moieties. These sulfonamides were assayed as inhibitors of two physiologically relevant isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), i.e., the cytosolic CA II (ubiquitous), and CA VII (brain-specific enzyme). Good CA inhibitory activity was detected for some of these derivatives, with inhibition constants (Ki) in the range of 17.5-863 nm against CA II; and 30-4200 nm against CA VII. PMID:19549076

  17. Phenolic compounds as antioxidants: carbonic anhydrase isoenzymes inhibitors.

    PubMed

    Gülçin, Ilhami; Beydemir, Şükrü

    2013-03-01

    Antioxidant compounds can scavenge free radicals and increase shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food, medicine and pharmaceutical products during processing and storage. An antioxidant molecule has been defined as any substance when found in low concentrations compared to that of an oxidizable substrate significantly delays or inhibits the oxidation. The major antioxidant compounds are especially phenolics and flavonoids, which are responsible for their health benefits. Carbonic anhydrase (EC 4.2.1.1., CA) is a pH regulatory/metabolic enzyme in all life kingdoms, being found in organisms all over the phylogenetic tree. It catalyzes the hydration of carbon dioxide (CO(2)) to bicarbonate (HCO(3) -) and the corresponding dehydration of HCO(3) -in acidic medium with regeneration of CO(2). Also, CA isoforms are found in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and electrolyte secretion. On the other hand, the phenyl moiety of phenol was found to lay in the hydrophobic part of the CA active site, where CO(2), the physiologic substrate of the CAs, binds in the precatalytic complex, explaining thus the behavior of phenol as a unique CO(2) competitive inhibitor. This review consists of two main sections. The first section is devoted to main phenolic antioxidant compounds in the foodstuffs and beverages. The second general section is about some definitions of CA inhibitory effects of the main phenolic compounds used for antioxidant activity. The phenolic compounds and acids had marked especially CA I and CA II inhibitory effects and might be used as leads for generating CA isoenzyme inhibitors. This class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs. In

  18. Why Is Carbonic Anhydrase Essential to Escherichia coli?

    PubMed Central

    Merlin, Christophe; Masters, Millicent; McAteer, Sean; Coulson, Andrew

    2003-01-01

    The can (previously yadF) gene of Escherichia coli encodes a β-class carbonic anhydrase (CA), an enzyme which interconverts CO2 and bicarbonate.Various essential metabolic processes require either CO2 or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO2 in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs. We calculate that demand for bicarbonate is 103- to 104-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO2 to bicarbonate is therefore necessary for growth. We find that can expression is ordinarily required for growth in air. It is dispensable if the atmospheric partial pressure of CO2 is high or during anaerobic growth in a closed vessel at low pH, where copious CO2 is generated endogenously. CynT, the single E. coli Can paralog, can, when induced with azide, replace Can; also, the γ-CA from Methanosarcina thermophila can at least partially replace it. Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated. However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities. Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress. PMID:14563877

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

    PubMed

    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.

  20. Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes.

    PubMed

    Arabaci, Betul; Gulcin, Ilhami; Alwasel, Saleh

    2014-01-01

    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. PMID:25014536

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

    PubMed

    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

  2. Carbonic anhydrase IX: regulation and role in cancer.

    PubMed

    Benej, Martin; Pastorekova, Silvia; Pastorek, Jaromir

    2014-01-01

    Tumor microenvironment substantially influences the process of tumorigenesis. In many solid tumors, imbalance between the demand of rapidly proliferating cancer cells and the capabilities of the vascular system generates areas with insufficient oxygen supply. In response to tumor hypoxia, cancer cells modulate their gene expression pattern to match the requirements of the altered microenvironment. One of the most significant adaptations to this milieu is the shift towards anaerobic glycolysis to keep up the energy demands. This oncogenic metabolism is often maintained also in aerobic cells. Lactic acid, its metabolic end-product, accumulates hand-in-hand with carbon dioxide, leading to acidification of the extracellular environment. Carbonic anhydrase IX (CA IX) is the most widely expressed gene in response to hypoxia. Its crucial role in intracellular pH maintenance represents the means by which cancer cells adapt to the toxic conditions of the extracellular milieu. Furthermore, the activity of CA IX stimulates the migratory pathways of cancer cells and is connected with the increase of the aggressive/invasive phenotype of tumors. CA IX expression in many types of tumors indicates its relevance as a general marker of tumor hypoxia. Moreover, its expression is closely related to prognosis of the clinical outcome in several tumor types. All above mentioned facts support the strong position of CA IX as a potential drug therapy target. Here, we summarize the state-of-the-art knowledge on its regulation and role in cancer development.

  3. Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes.

    PubMed

    Arabaci, Betul; Gulcin, Ilhami; Alwasel, Saleh

    2014-01-01

    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. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

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

    PubMed

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

    2014-06-01

    This study examined the possible involvement of carbonic anhydrase activation in response to an endometriosis-related increase in oxidative stress. Peripheral blood samples obtained from 27 healthy controls and 30 endometriosis patients, classified as having endometriosis by histological examination of surgical specimens, were analysed by multiple immunoassay and carbonic anhydrase activity assay. Red blood cells (RBC) were analysed for glutathionylated protein (GSSP) content in the membrane, total glutathione (GSH) in the cytosol and carbonic anhydrase concentration and activity. In association with a membrane increase of GSSP and a cytosolic decrease of GSH content in endometriosis patients, carbonic anhydrase significantly increased (P < 0.0001) both monomerization and activity compared with controls. This oxidation-induced activation of carbonic anhydrase was positively and significantly correlated with the GSH content of RBC (r = 0.9735, P < 0.001) and with the amount of the 30-kDa monomer of carbonic anhydrase (r = 0.9750, P < 0.001). Because carbonic anhydrase activation is implied in many physiological and biochemical processes linked to pathologies such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis. These data open promising working perspectives for diagnosis and treatment of endometriosis and hopefully of other oxidative stress-related diseases. Endometriosis is a chronic disease associated with infertility and local inflammatory response, which is thought to spread rapidly throughout the body as a systemic subclinical inflammation. One of the causes in the pathogenesis/evolution of endometriosis is oxidative stress, which occurs when reactive oxygen species are produced faster than the endogenous antioxidant defence systems can neutralize them. Once produced, reactive oxygen species can alter the morphological and functional properties of endothelial cells, including

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

    NASA Astrophysics Data System (ADS)

    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

  7. Circadian periodicity in salivary carbonic anhydrase VI concentration.

    PubMed

    Parkkila, S; Parkkila, A K; Rajaniemi, H

    1995-06-01

    Carbonic anhydrase VI (CA VI) is secreted into the saliva by the serous acinar cells of the parotid and submandibular glands. Saliva samples from six healthy male volunteers were analysed for concentrations of CA VI throughout the 24 h period by means of a specific time-resolved immunofluorometric assay and the levels were compared with amylase activity. The sleeping period was from 00.10 h to 07.30 h and the subjects had breakfast at 07.30 h and regular meals at 13.30 h and 19.30 h. Saliva secretion decreased markedly during the sleeping period in all the subjects except one. The levels of both CA VI and amylase activity varied greatly among the subjects, but in a parallel manner, and declined to a very low level during the sleeping period. Dexamethasone intake at midnight had no effect on the morning rise in either enzyme. When the sleeping period was postponed from 06.10 h to 11.30 h both enzyme concentrations declined during the night and continued to be low until the subjects awoke at 11.30 h, whereas salivary secretion was low only during the sleeping period. Our results suggest that CA VI secretion follows a circadian periodicity that is comparable to amylase secretion but independent of salivary secretion.

  8. Structure-based drug discovery of carbonic anhydrase inhibitors.

    PubMed

    Supuran, Claudiu T

    2012-12-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA; EC 4.2.1.1) has pharmacologic applications in the field of anti-glaucoma, anti-convulsant and anti-cancer agents. But recently, it has also emerged that these enzymes have the potential for designing anti-infective drugs (anti-fungal and anti-bacterial agents) with a novel mechanism of action. Sulphonamides and their isosteres (sulphamates/sulphamides) constitute the main class of CA inhibitors (CAIs), which bind to the metal ion from the enzyme active site. Recently, the dithiocarbamates (DTCs), possessing a similar mechanism of action, were reported as a new class of inhibitors. These types of CAIs will be discussed in detail in this review. Novel drug design strategies have been reported ultimately based on the tail approach for obtaining sulphonamides/DTCs, 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. Most of the promising data have been obtained by combining x-ray crystallography of enzyme-inhibitor adducts with novel synthetic approaches for generating chemical diversity. Whereas sulphonamide - NO donating hybrid drugs were reported as effective anti-glaucoma agents, most of the interesting new inhibitors were designed for inhibiting specifically the tumour-associated isoforms CA IX and XII, validated targets for imaging and treatment of hypoxic tumours. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the DTC and carboxylate types, will be also reviewed. PMID:22468747

  9. Glaucoma and the applications of carbonic anhydrase inhibitors.

    PubMed

    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

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

    PubMed

    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

  11. New natural product carbonic anhydrase inhibitors incorporating phenol moieties.

    PubMed

    Karioti, Anastasia; Ceruso, Mariangela; Carta, Fabrizio; Bilia, Anna-Rita; Supuran, Claudiu T

    2015-11-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. The need to find selective CA inhibitors (CAIs) triggered the investigation of natural product libraries, which proved to be a valid source of agents with such an activity, as demonstrated for the phenols, polyamines and coumarins. Herein we report an in vitro inhibition study of human (h) CA isoforms hCAs I, II, IV, VII and XII with a panel of natural polyphenols including flavones, flavonols, flavanones, flavanols, isoflavones and depsides, some of which extracted from Quercus ilex and Salvia miltiorrhiza. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting some important isoforms over the off-target ones hCA I and II.

  12. Carbonic anhydrase enzymes regulate mast cell-mediated inflammation.

    PubMed

    Henry, Everett K; Sy, Chandler B; Inclan-Rico, Juan M; Espinosa, Vanessa; Ghanny, Saleena S; Dwyer, Daniel F; Soteropoulos, Patricia; Rivera, Amariliz; Siracusa, Mark C

    2016-08-22

    Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2-associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy-like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell-mediated inflammation. PMID:27526715

  13. Carbonic anhydrase and acid-base regulation in fish.

    PubMed

    Gilmour, K M; Perry, S F

    2009-06-01

    Carbonic anhydrase (CA) is the zinc metalloenzyme that catalyses the reversible reactions of CO(2) with water. CA plays a crucial role in systemic acid-base regulation in fish by providing acid-base equivalents for exchange with the environment. Unlike air-breathing vertebrates, which frequently utilize alterations of breathing (respiratory compensation) to regulate acid-base status, acid-base balance in fish relies almost entirely upon the direct exchange of acid-base equivalents with the environment (metabolic compensation). The gill is the critical site of metabolic compensation, with the kidney playing a supporting role. At the gill, cytosolic CA catalyses the hydration of CO(2) to H(+) and HCO(3)(-) for export to the water. In the kidney, cytosolic and membrane-bound CA isoforms have been implicated in HCO(3)(-) reabsorption and urine acidification. In this review, the CA isoforms that have been identified to date in fish will be discussed together with their tissue localizations and roles in systemic acid-base regulation.

  14. Carbonic anhydrase and the molecular evolution of C4 photosynthesis.

    PubMed

    Ludwig, Martha

    2012-01-01

    C(4) photosynthesis, a biochemical CO(2)-concentrating mechanism (CCM), evolved more than 60 times within the angiosperms from C(3) ancestors. The genus Flaveria, which contains species demonstrating C(3), C(3)-C(4), C(4)-like or C(4) photosynthesis, is a model for examining the molecular evolution of the C(4) pathway. Work with carbonic anhydrase (CA), and C(3) and C(4) Flaveria congeners has added significantly to the understanding of this process. The C(4) form of CA3, a β-CA, which catalyses the first reaction in the C(4) pathway by hydrating atmospheric CO(2) to bicarbonate in the cytosol of mesophyll cells (mcs), evolved from a chloroplastic C(3) ancestor. The molecular modifications to the ancestral CA3 gene included the loss of the sequence encoding the chloroplast transit peptide, and mutations in regulatory regions that resulted in high levels of expression in the C(4) mesophyll. Analyses of the CA3 proteins and regulatory elements from Flaveria photosynthetic intermediates indicated C(4) biochemistry very likely evolved in a specific, stepwise manner in this genus. The details of the mechanisms involved in the molecular evolution of other C(4) plant β-CAs are unknown; however, comparative genetics indicate gene duplication and neofunctionalization played significant roles as they did in Flaveria.

  15. Proton Transport in Carbonic Anhydrase: Insights from Molecular Simulation

    PubMed Central

    Maupin, C. Mark; Voth, Gregory A.

    2009-01-01

    Summary This article reviews the insights gained from molecular simulations of human carbonic anhydrase II (HCA II) utilizing non-reactive and reactive force fields. The simulations with a reactive force field explore protein transfer and transport via Grotthuss shuttling, while the non-reactive simulations probe the larger conformational dynamics that underpin the various contributions to the rate-limiting proton transfer event. Specific attention is given to the orientational stability of the His64 group and the characteristics of the active site water cluster, in an effort to determine both of their impact on the maximal catalytic rate. The explicit proton transfer and transport events are described by the multistate empirical valence bond (MS-EVB) method, as are alternative pathways for the excess proton charge defect to enter/leave the active site. The simulation results are interpreted in light of experimental results on the wild-type enzyme and various site-specific mutations of HCA II in order to better elucidate the key factors that contribute to its exceptional efficiency. PMID:19765680

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

    PubMed

    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.

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

    SciTech Connect

    Henry, R.P.

    1987-05-01

    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 approx. 15 ..mu..M; 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 /sup 18/O-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.

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

    PubMed

    Henry, R P

    1987-05-01

    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 Ki values of approximately 15 microM; 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 or in vitro in the study of membrane-associated CA.

  19. Characterization of carbonic anhydrase-inhibitor noncovalent complexes

    SciTech Connect

    Cheng, Xueheng; Chen, R.; Bruce, J.E.

    1995-12-31

    Competitive binding of two mixtures of inhibitors to bovine carbonic anhydrase H (BCAII) was studied using electrospray ionization mass spectrometry (ESI-MS). The first mixture contained inhibitors with hydrocarbon/fluorohydrocarbon linker groups and with an 800 fold span of binding constants. The second contained inhibitors with dipeptide extensions synthesized using the solid phase method. Noncovalent enzyme-inhibitor complexes were observed from solutions in 10 mM ammonia acetate having abundances consistent with their relative binding constants measured in solution. The inhibitor with highest affinity was readily identified in an equimolar mixture. The inhibitors with very low affinity were identified to form specific complexes as well. Several control experiments including acidifying the solution or removing the Zn metal from the enzyme resulted in the disappearance of the enzyme-inhibitor complexes, (mass spectrometrically) observed complexation and characterization of biomolecular binding. Good correlation between gas phase inhibitor ion abundances and their binding constants in solution were observed. Structural information and relative binding constants of masses were obtained using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) through multi-stage dissociation experiments. These results support the role of ESI-FTICR-MS in the study of specific noncovalent associations from solution, and show that its unique capabilities can be exploited to extend studies to large mixtures of inhibitors in drug leads discovery.

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

    PubMed

    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

  1. Function of carbonic anhydrase IX in glioblastoma multiforme.

    PubMed

    Proescholdt, Martin A; Merrill, Marsha J; Stoerr, Eva-Maria; Lohmeier, Annette; Pohl, Fabian; Brawanski, Alexander

    2012-11-01

    Carbonic anhydrase (CA) IX is over-expressed in glioblastoma; however, its functions in this context are unknown. Metabolically, glioblastomas are highly glycolytic, leading to a significant lactic acid load. Paradoxically, the intracellular pH is alkaline. We hypothesized that CAIX contributes to the extrusion of hydrogen ions into the extracellular space, thereby moderating intra- and extracellular pH and creating an environment conductive to enhanced invasion. We investigated the role of CAIX as a prognostic marker in patients with glioblastoma and its biological function in vitro. CAIX expression was analyzed in 59 patients with glioblastoma by immunohistochemistry. The expression levels were correlated to overall survival. In vitro, U251 and Ln 18 glioblastoma cells were incubated under hypoxia to induce CAIX expression, and RNA interference (RNAi) was used to examine the function of CAIX on cell attachment, invasion, intracellular energy transfer, and susceptibility to adjuvant treatment. High CAIX expression was identified as an independent factor for poor survival in patients with glioblastoma. In vitro, cell attachment and invasion were strongly reduced after knockdown of CAIX. Finally, the effects of radiation and chemotherapy were strongly augmented after CAIX interference and were accompanied by a higher rate of apoptotic cell death. CAIX is an independent prognostic factor for poor outcome in patients with glioblastoma. Cell attachment, invasion, and survival during adjuvant treatment are significantly influenced by high CAIX expression. These results indicate that inhibition of CAIX is a potential metabolic target for the treatment of patients with glioblastoma. PMID:23074198

  2. Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland.

    PubMed Central

    Fernley, R T; Coghlan, J P; Wright, R D

    1988-01-01

    Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus. Images Fig. 4. Fig. 6. Fig. 7. PMID:3124821

  3. IgM natural autoantibodies against bromelain-treated mouse red blood cells recognise carbonic anhydrase.

    PubMed

    Jonusys, A M; Cox, K O; Steele, E J

    1991-01-01

    Carbonic anhydrase (CA) from mouse erythrocyte membranes is recognised as an autoantigen in Western blotting experiments with FUB 1, a murine IgM monoclonal antibody that binds both phosphatidylcholine and bromelain-treated mouse red blood cells (BrMRBC). Serum from mice stimulated with lipopolysaccharide (LPS-serum) also recognises CA. From SDS-PAGE, and blotting experiments with whole mouse erythrocytes, we found two closely spaced glycoprotein bands in the 30 kD region that reacted with both FUB 1 and LPS-serum. One of the molecular weight markers, bovine carbonic anhydrase which is of a molecular weight of about 30 kD, electrophoresed in the same 30 kD region also reacted with these antibodies. Carbonic anhydrases from a range of mammalian species were found to be crossreactive with FUB 1 and LPS-serum by Western blotting, whereas human glycophorin A and human asialoglycophorin were not recognised by the antibodies. FUB 1 specifically recognises both native and denatured bovine carbonic anhydrase in ELISA assays. The serological identity of the determinants of CA and BrMRBC was confirmed by specific absorption of both FUB 1 and LPS-serum with BrMRBC and normal mouse erythrocytes. We propose that a native autoantigenic epitope on erythrocytes may be revealed by the proteolytic action of bromelain and that this determinant is associated, at least in part, with carbonic anhydrase.

  4. Carbonic anhydrases and anion transport in mosquito midgut pH regulation

    PubMed Central

    Linser, Paul J.; Smith, Kristin E.; Seron, Terri J.; Neira Oviedo, Marco

    2009-01-01

    Summary Mosquito larvae use a digestive strategy that is relatively rare in nature. The anterior half of the larval mosquito midgut has a luminal pH that ranges between 10.5 and 11.5. Most other organisms, both large and small, initiate digestion in an acid medium. The relative uniqueness of the highly alkaline digestive strategy has been a long-standing research focus in larval lepidopterans. More recently, the disease vector potential of mosquitoes has fueled specific interest in larval mosquito biology and the alkaline digestive environment in the midgut. The probable principle anion influencing the highly alkaline gut lumen is bicarbonate/carbonate. Bicarbonate/carbonate is regulated at least in part by the activity of carbonic anhydrases. Hence, we have focused attention on the carbonic anhydrases of the mosquito larva. Anopheles gambiae, the major malaria mosquito of Africa, is an organism with a published genome which has facilitated molecular analyses of the 12 carbonic anhydrase genes annotated for this mosquito. Microarray expression analyses, tissue-specific quantitative RT-PCR, and antibody localization have been used to generate a picture of carbonic anhydrase distribution in the larval mosquito. Cytoplasmic, GPI-linked extracellular membrane-bound and soluble extracellular carbonic anhydrases have been located in the midgut and hindgut. The distribution of the enzymes is consistent with an anion regulatory system in which carbonic anhydrases provide a continuous source of bicarbonate/carbonate from the intracellular compartments of certain epithelial cells to the ectoperitrophic space between the epithelial cells and the acellular membrane separating the food bolus from the gut cells and finally into the gut lumen. Carbonic anhydrase in specialized cells of the hindgut (rectum) probably plays a final role in excretion of bicarbonate/carbonate into the aquatic environment of the larva. Detection and characterization of classic anion exchangers of the

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

    PubMed

    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

  6. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

    PubMed

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

    2016-01-01

    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

  7. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    PubMed Central

    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

  8. Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage

    PubMed Central

    Bejaoui, Mohamed; Pantazi, Eirini; De Luca, Viviana; Panisello, Arnau; Folch-Puy, Emma; Hotter, Georgina; Capasso, Clemente; T. Supuran, Claudiu; Rosselló-Catafau, Joan

    2015-01-01

    Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation. PMID:26225852

  9. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides.

    PubMed

    Ozensoy, Ozen; Arslan, Mikail; Supuran, Claudiu T

    2011-10-01

    A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO(2) hydration reaction were measured. With a k(cat)/K(m) of 1.1 × 10(8) M(-1) s(-1), and a k(cat) of 1.3 × 10(6) s(-1), clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K(I)s in the range of 1.9-3460 nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme. PMID:21476925

  13. Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids.

    PubMed

    Vullo, Daniela; Del Prete, Sonia; Capasso, Clemente; Supuran, Claudiu T

    2016-03-01

    The β-carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3μM) but more effectively activated by d-Phe, l-DOPA, l-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine, with KAs of 5.82-10.9μM. The best activators were l-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81μM. This study may help a better understanding of the activation mechanisms of β-CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents.

  14. Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids.

    PubMed

    Vullo, Daniela; Del Prete, Sonia; Capasso, Clemente; Supuran, Claudiu T

    2016-03-01

    The β-carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3μM) but more effectively activated by d-Phe, l-DOPA, l-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine, with KAs of 5.82-10.9μM. The best activators were l-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81μM. This study may help a better understanding of the activation mechanisms of β-CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents. PMID:26856923

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

    PubMed Central

    Asahi, Masumi G; Bobarnac Dogaru, Gabriela L; Onishi, Spencer M; Gallemore, Ron P

    2015-01-01

    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. PMID:26664246

  16. Carbonic Anhydrase II Deficiency in a Saudi Woman

    PubMed Central

    Alhuzaim, Omar N; Almohareb, Ohoud M; Sherbeeni, Safiya M

    2015-01-01

    OBJECTIVE Carbonic anhydrase (CA) II deficiency is a rare autosomal recessive disorder caused by mutation in the CA II gene that leads to osteopetrosis, renal tubular acidosis (RTA), and cerebral calcification. Our aim is to present a patient with the classic triad of CA II deficiency syndrome to enhance the awareness about this rare syndrome. METHODS We describe the clinical and radiological findings of a Saudi woman patient with CA II deficiency syndrome. RESULTS A Saudi woman in her 20s presented to our hospital for evaluation of increased bone density. She was known to have delayed developmental milestone with growth retardation and poor scholastic performance. She had multiple fragile fractures started at the age of 15 involving the lower extremities. A physical examination revealed dysmorphic features and intellectual disability with intelligence quotient (IQ) of 36. The initial blood workup showed a picture of distal RTA with hypokalemia, and the radiological imaging confirmed the presence of osteopetrosis and multiple kidney stones. The combination of osteopetrosis with RTA raised the possibility of CA II deficiency. Therefore, computed tomography (CT) of the brain was done and showed intracranial calcification involving the basal ganglia. She was started on potassium chloride and sodium bicarbonate. In addition, she underwent right-sided percutaneous nephrolithotripsy. Her DNA analysis came to show a sequence variant c.232+1G>A, which was detected in both of the CA II genes (homozygous). CONCLUSION Early recognition of the disease is a key, as an early appropriate treatment institution is essential in order to prevent further complications. PMID:25674028

  17. Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

    PubMed Central

    Alvarez, Bernardo V; Johnson, Danielle E; Sowah, Daniel; Soliman, Daniel; Light, Peter E; Xia, Ying; Karmazyn, Morris; Casey, Joseph R

    2007-01-01

    Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+–H+ exchanger (NHE1) and Cl−–HCO3− exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3−, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 ± 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50 = 18 μm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 ± 0.3 μm and 2.7 ± 0.3 μm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 ± 0.4 Hz to 0.77 ± 0.04 Hz following 24 h treatment; these Ca2+-handling abnormalities were completely prevented by ETZ (0.28 ± 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype. PMID:17124262

  18. The molecular evolution of β-carbonic anhydrase in Flaveria.

    PubMed

    Ludwig, Martha

    2011-05-01

    Limited information exists regarding molecular events that occurred during the evolution of C(4) plants from their C(3) ancestors. The enzyme β-carbonic anhydrase (CA; EC 4.2.1.1), which catalyses the reversible hydration of CO(2), is present in multiple forms in C(3) and C(4) plants, and has given insights into the molecular evolution of the C(4) pathway in the genus Flaveria. cDNAs encoding three distinct isoforms of β-CA, CA1-CA3, have been isolated and examined from Flaveria C(3) and C(4) congeners. Sequence data, expression analyses of CA orthologues, and chloroplast import assays with radiolabelled CA precursor proteins from the C(3) species F. pringlei Gandoger and the C(4) species F. bidentis (L.) Kuntze have shown that both contain chloroplastic and cytosolic forms of the enzyme, and the potential roles of these isoforms are discussed. The data also identified CA3 as the cytosolic isoform important in C(4) photosynthesis and indicate that the C(4) CA3 gene evolved as a result of gene duplication and neofunctionalization, which involved mutations in coding and non-coding regions of the ancestral C(3) CA3 gene. Comparisons of the deduced CA3 amino acid sequences from Flaveria C(3), C(4), and photosynthetic intermediate species showed that all the C(3)-C(4) intermediates investigated and F. brownii, a C(4)-like species, have a C(3)-type CA3, while F. vaginata, another C(4)-like species, contains a C(4)-type CA3. These observations correlate with the photosynthetic physiologies of the intermediates, suggesting that the molecular evolution of C(4) photosynthesis in Flaveria may have resulted from a temporally dependent, stepwise modification of protein-encoding genes and their regulatory elements.

  19. A carbonic anhydrase from the archaeon Methanosarcina thermophila.

    PubMed Central

    Alber, B E; Ferry, J G

    1994-01-01

    Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila was purified > 10,000-fold (22% recovery) to apparent homogeneity with a specific activity of 4872 units/mg. The estimated native molecular mass of the enzyme is 84 kDa based on gel filtration chromatography. SDS/PAGE revealed one protein band with an apparent molecular mass of 40 kDa. The M. thermophila CA is less sensitive than human CA isozyme II toward inhibition by sulfonamides and monovalent ions. The gene encoding this CA was cloned into pUC18 and sequenced. Escherichia coli harboring the recombinant plasmid expresses CA activity (2.3 units/mg of cell extract protein). Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The calculated molecular mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates the subunit size and that the native enzyme is a tetramer. To our knowledge, the deduced amino acid sequence has no significant identity to any known CA but has 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO2-concentrating-mechanism protein from Synechococcus PCC7942 and 28% sequence identity to the deduced sequence of ferripyochelin binding protein from Pseudomonas aeruginosa. Thus, our results indicate that this archaeal CA represents a distinct class of CAs and provide a basis to determine physiological roles for CA in acetotrophic anaerobes. Images PMID:8041719

  20. Dissolved carbonic anhydrase for enhancing post-combustion carbon dioxide hydration in aqueous ammonia

    SciTech Connect

    Collett, James R.; Heck, Robert W.; Zwoster, Andy

    2011-04-01

    Aqueous ammonia solvents that capture CO2 as ionic complexes of carbonates with ammonium have recently been advanced as alternatives to amine-based solvents due to their lower energy requirements for thermal regeneration. In ammonia based solvents, the hydration of CO2 to form bicarbonate may become a rate-limiting step as the CO2 loading increases and the resulting pH level of the solvent decreases. Variants of the enzyme carbonic anhydrase can accelerate the reversible hydration of CO2 to yield bicarbonate by more than 10(6)-fold. The possible benefit of bovine carbonic anhydrase (BCA) addition to solutions of aqueous ammonia to enhance CO2 hydration was investigated in semi-batch reactions within continuously stirred tank reactors or in a bubble column gas-liquid contactor. Adding 154 mg/liter of BCA to 2 M aqueous ammonia provided a 34.1% overall increase in the rate of CO2 hydration (as indicated by the production of [H+]) as the pH declined from 9.6 to 8.6 during sparging with a 15% CO2, 85% N-2 gas at a flow rate of 3 lpm. The benefits of adding BCA to enhance CO2 hydration were only discernable below similar to pH 9. The implications of the apparent pH limitations on the utility of BCA are discussed in the context of absorber unit operation design. Possible embodiments of carbonic anhydrase as either an immobilized catalyst or as a dissolved, recirculating catalyst in potential plant scale aqueous ammonia systems are considered as well. (C) 2010 Published by Elsevier Ltd.

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

    SciTech Connect

    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.

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

    PubMed

    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

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

    PubMed Central

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

    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 groups for enzyme immobilization. XPS and a colorimetric amine assay confirmed higher amine densities on the chitosan coated fiber compared to control fiber. Chitosan/CA coated fibers exhibited accelerated CO2 removal in scaled-down gas exchange devices in buffer and blood (115 % enhancement vs. control, 37 % enhancement vs. control, respectively). Carbonic anhydrase immobilized directly on hollow fiber membranes without chitosan tethering resulted in no enhancement in CO2 removal. Additionally, fibers coated with chitosan/carbonic anhydrase demonstrated reduced platelet adhesion when exposed to blood compared to control and heparin coated fibers. PMID:23888352

  4. Thienothiopyransulfonamides as Complexing Agents for the Preparation of Dual Carbonic Anhydrase Inhibitors

    PubMed Central

    Supuran, Claudiu T.

    1995-01-01

    Co(II); Zn(II) and Cu(II) complexes of two new sulfonamide carbonic anhydrase (CA) inhibitors, derivatives of thienothiopyran-2-sulfonamide, were prepared and characterized by analytic, spectroscopic, magnetic and conductimetric measurements. The new complexes are more potent CA inhibitors than the parent sulfonamides, with IC50 values around 0.1 nM, against isozyme CA II. PMID:18472783

  5. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... system. 866.5200 Section 866.5200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5200 Carbonic anhydrase B and C immunological test system. (a) Identification. A...

  6. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... system. 866.5200 Section 866.5200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5200 Carbonic anhydrase B and C immunological test system. (a) Identification. A...

  7. Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

    PubMed

    Kozliak, E I; Guilloton, M B; Gerami-Nejad, M; Fuchs, J A; Anderson, P M

    1994-09-01

    Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.

  8. Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

    PubMed Central

    Kozliak, E I; Guilloton, M B; Gerami-Nejad, M; Fuchs, J A; Anderson, P M

    1994-01-01

    Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate. Images PMID:8083164

  9. Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

    PubMed Central

    Alvizo, Oscar; Nguyen, Luan J.; Savile, Christopher K.; Bresson, Jamie A.; Lakhapatri, Satish L.; Solis, Earl O. P.; Fox, Richard J.; Broering, James M.; Benoit, Michael R.; Zimmerman, Sabrina A.; Novick, Scott J.; Liang, Jack; Lalonde, James J.

    2014-01-01

    Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction. PMID:25368146

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

    SciTech Connect

    Weiwei, Zhang; Hu, Renming

    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.

  11. A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

    PubMed Central

    Guilloton, M B; Lamblin, A F; Kozliak, E I; Gerami-Nejad, M; Tu, C; Silverman, D; Anderson, P M; Fuchs, J A

    1993-01-01

    Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and

  12. A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

    PubMed

    Guilloton, M B; Lamblin, A F; Kozliak, E I; Gerami-Nejad, M; Tu, C; Silverman, D; Anderson, P M; Fuchs, J A

    1993-03-01

    Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and

  13. Sequestration of carbon dioxide by the hydrophobic pocket of the carbonic anhydrases.

    PubMed

    Domsic, John F; McKenna, Robert

    2010-02-01

    The interaction between carbon dioxide (CO(2)) and the alpha-class carbonic anhydrase, human CA 2 (HCA2) exists for only a short period due to the rapid catalytic turnover by this enzyme. The fleeting nature of this interaction has led to difficulties in its direct analysis, with previous studies placing the CO(2) in the hydrophobic pocket of HCA2's active site. A more precise location was determined via the crystal structure of CO(2) trapped in both wild-type (holo) and zinc-free (apo) HCA2. This provided a detailed description of the means by which CO(2) is held and orientated for optimal catalysis. This information can be extended to the beta and gamma class enzymes to help elucidate the binding mode of CO(2) in these enzymes. PMID:19679198

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

    PubMed

    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

  15. Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice.

    PubMed

    Jilka, R L; Rogers, J I; Khalifah, R G; Vaananen, H K

    1985-01-01

    The content of carbonic anhydrase isozymes I (CA I) and II (CA II) in red blood cells and bone of osteopetrotic microphthalmic (mi/mi) mice was analyzed. Monospecific rabbit polyclonal antibodies against purified rat carbonic anhydrase I or II detected both isozymes in hemolysates of both normal and mi/mi mice. Total carbonic anhydrase activity measurements of hemolysates from normal or mi/mi mice were identical. A procedure based on bromopyruvate inactivation was devised to measure the relative contributions of CA I and II isozymes to the carbon dioxide hydration activity of hemolyzates. CA II dominates the observed activity of hemolysates of normal and mi/mi mice. Immunohistochemical studies showed that CA II was present in osteoclasts of tibial and calvarial bones of both normal and mi/mi mice. Thus, in contrast to the several cases of inherited osteopetrosis in humans, there is no lack of active CA II in erythrocytes of mi/mi mice. The absence of CA II, therefore, does not appear to play a role in the etiology of osteopetrosis in the mi/mi mouse.

  16. Free-solution interaction assay of carbonic anhydrase to its inhibitors using back-scattering interferometry.

    PubMed

    Morcos, Ereny F; Kussrow, Amanda; Enders, Carolyn; Bornhop, Darryl

    2010-11-01

    Back-scattering interferometry (BSI) is a label-free, free-solution, small-volume technique used for characterizing binding interactions, which is also relevant to a growing number of biosensing applications including drug discovery. Here, we use BSI to characterize the interaction of carbonic anhydrase enzyme II with five well-known carbonic anhydrase enzyme II inhibitors (± sulpiride, sulfanilamide, benzene sulfonamide, dansylamide, and acetazolamide) in the presence of DMSO. Dissociation constants calculated for each interaction were consistent with literature values previously obtained using surface plasmon resonance and fluorescence-based competition assays. Results demonstrate the potential of BSI as a drug-screening tool which is fully compatible with DMSO and does not require immobilization or labeling, therefore allowing binding interactions to be characterized in the native state. BSI has the potential for reducing labor costs, sample consumption, and assay time while providing enhanced reliability over existing techniques. PMID:20972990

  17. Synthesis and carbonic anhydrase inhibitory properties of 1,3-dicarbonyl derivatives of methylaminobenzene-sulfonamide.

    PubMed

    Demirci, Tuna; Arslan, Mustafa; Bilen, Çiğdem; Demir, Dudu; Gençer, Nahit; Arslan, Oktay

    2014-02-01

    Abstract 1,3-Dicarbonyl derivatives of methylaminobenzene-sulfonamide were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and hCA II were evaluated. hCA I and hCA II from human erythrocytes were purified by a simple one-step procedure by using Sepharose 4B-L-tyrosine-sulfanilamide affinity column. Our results show that the synthesized compounds inhibited the activity of carbonic anhydrase (CA) I and CA II. Among them, 2b and 2e were found to be the most active (IC50=2.12 and 2.52 µM) for hCA I and hCA II, respectively. PMID:23356427

  18. Co-production of carbonic anhydrase and phycobiliproteins by Spirulina sp. and Synechococcus nidulans.

    PubMed

    Ores, Joana da Costa; Amarante, Marina Campos Assumpção de; Kalil, Susana Juliano

    2016-11-01

    The aim of this work was to study the co-production of the carbonic anhydrase, C-phycocyanin and allophycocyanin during cyanobacteria growth. Spirulina sp. LEB 18 demonstrated a high potential for simultaneously obtaining the three products, achieving a carbonic anhydrase (CA) productivity of 0.97U/L/d and the highest C-phycocyanin (PC, 5.9μg/mL/d) and allophycocyanin (APC, 4.3μg/mL/d) productivities. In the extraction study, high extraction yields were obtained from Spirulina using an ultrasonic homogenizer (CA: 25.5U/g; PC: 90mg/g; APC: 70mg/g). From the same biomass, it was possible to obtain three biomolecules that present high industrial value. PMID:27494103

  19. Co-production of carbonic anhydrase and phycobiliproteins by Spirulina sp. and Synechococcus nidulans.

    PubMed

    Ores, Joana da Costa; Amarante, Marina Campos Assumpção de; Kalil, Susana Juliano

    2016-11-01

    The aim of this work was to study the co-production of the carbonic anhydrase, C-phycocyanin and allophycocyanin during cyanobacteria growth. Spirulina sp. LEB 18 demonstrated a high potential for simultaneously obtaining the three products, achieving a carbonic anhydrase (CA) productivity of 0.97U/L/d and the highest C-phycocyanin (PC, 5.9μg/mL/d) and allophycocyanin (APC, 4.3μg/mL/d) productivities. In the extraction study, high extraction yields were obtained from Spirulina using an ultrasonic homogenizer (CA: 25.5U/g; PC: 90mg/g; APC: 70mg/g). From the same biomass, it was possible to obtain three biomolecules that present high industrial value.

  20. Sulfa drugs as inhibitors of carbonic anhydrase: new targets for the old drugs.

    PubMed

    al-Rashida, Mariya; Hussain, Sajad; Hamayoun, Mehwish; Altaf, Aisha; Iqbal, Jamshed

    2014-01-01

    Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR'). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a-5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b-5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds.

  1. Discovery of Potent Carbonic Anhydrase and Acetylcholinesterase Inhibitors: 2-Aminoindan β-Lactam Derivatives

    PubMed Central

    Genç, Hayriye; Kalin, Ramazan; Köksal, Zeynep; Sadeghian, Nastaran; Kocyigit, Umit M.; Zengin, Mustafa; Gülçin, İlhami; Özdemir, Hasan

    2016-01-01

    β-Lactams are pharmacologically important compounds because of their various biological uses, including antibiotic and so on. β-Lactams were synthesized from benzylidene-inden derivatives and acetoxyacetyl chloride. The inhibitory effect of these compounds was examined for human carbonic anhydrase I and II (hCA I, and II) and acetylcholinesterase (AChE). The results reveal that β-lactams are inhibitors of hCA I, II and AChE. The Ki values of β-lactams (2a–k) were 0.44–6.29 nM against hCA I, 0.93–8.34 nM against hCA II, and 0.25–1.13 nM against AChE. Our findings indicate that β-lactams (2a–k) inhibit both carbonic anhydrases (CA) isoenzymes and AChE at low nanomolar concentrations. PMID:27775608

  2. Sulfa Drugs as Inhibitors of Carbonic Anhydrase: New Targets for the Old Drugs

    PubMed Central

    al-Rashida, Mariya; Hussain, Sajad; Hamayoun, Mehwish; Altaf, Aisha

    2014-01-01

    Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR′). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a–5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b–5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds. PMID:25538942

  3. Sulfa drugs as inhibitors of carbonic anhydrase: new targets for the old drugs.

    PubMed

    al-Rashida, Mariya; Hussain, Sajad; Hamayoun, Mehwish; Altaf, Aisha; Iqbal, Jamshed

    2014-01-01

    Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR'). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a-5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b-5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds. PMID:25538942

  4. CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor.

    PubMed

    Cudennec, Benoit; Rousseau, Marthe; Lopez, Evelyne; Fouchereau-Peron, Martine

    2006-11-01

    The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.

  5. Carbonic anhydrase activity in the red blood cells of sea level and high altitude natives.

    PubMed

    Gamboa, J; Caceda, R; Gamboa, A; Monge-C, C

    2000-01-01

    Red blood cell carbonic anhydrase (CA) activity has not been studied in high altitude natives. Because CA is an intraerythocytic enzyme and high altitude natives are polycythemic, it is important to know if the activity of CA per red cell volume is different from that of their sea level counterparts. Blood was collected from healthy subjects living in Lima (150m) and from twelve subjects from Cerro de Pasco (4330m), and hematocrit and carbonic anhydrase activity were measured. As expected, the high altitude natives had significantly higher hematocrits than the sea level controls (p = 0.0002). No difference in the CA activity per milliliter of red cells was found between the two populations. There was no correlation between the hematocrit and CA activity.

  6. Borate-catalyzed carbon dioxide hydration via the carbonic anhydrase mechanism.

    PubMed

    Guo, Dongfang; Thee, Hendy; da Silva, Gabriel; Chen, Jian; Fei, Weiyang; Kentish, Sandra; Stevens, Geoffrey W

    2011-06-01

    The hydration of CO(2) plays a critical role in carbon capture and geoengineering technologies currently under development to mitigate anthropogenic global warming and in environmental processes such as ocean acidification. Here we reveal that borate catalyzes the conversion of CO(2) to HCO(3)(-) via the same fundamental mechanism as the enzyme carbonic anhydrase, which is responsible for CO(2) hydration in the human body. In this mechanism the tetrahydroxyborate ion, B(OH)(4)(-), is the active form of boron that undergoes direct reaction with CO(2). In addition to being able to accelerate CO(2) hydration in alkaline solvents used for carbon capture, we hypothesize that this mechanism controls CO(2) uptake by certain saline bodies of water, such as Mono Lake (California), where previously inexplicable influx rates of inorganic carbon have created unique chemistry. The new understanding of CO(2) hydration provided here should lead to improved models for the carbon cycle in highly saline bodies of water and to advances in carbon capture and geoengineering technology.

  7. Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

    SciTech Connect

    Tu, C.K.; Paranawithana, S.R.; Jewell, D.A.; Tanhauser, S.M.; LoGrasso, P.V.; Wynns, G.C.; Laipis, P.J.; Silverman, D.N. )

    1990-07-10

    Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.

  8. Specific immunohistochemical pattern of carbonic anhydrase IX is helpful for the diagnosis of CNS hemangioblastoma.

    PubMed

    Schaller, Tina; Bode, Markus; Berlis, Ansgar; Frühwald, Michael C; Lichtmannegger, Ines; Endhardt, Katharina; Märkl, Bruno

    2015-07-01

    Hemangioblastomas are rare capillary-rich tumors predominantly found in the CNS. The histological appearance of these tumors varies across a broad spectrum. Several entities show considerable histomorphological similarities to hemangioblastomas. Therefore, morphological evaluation can be challenging. In this study, we evaluated the diagnostic utility of immunohistochemistry using antibodies against carbonic anhydrase IX and cytokeratin staining. Within our files, we identified 20 hemangioblastomas. A collection of 46 other tumors relevant to the differential diagnosis (12 pilocytic astrocytomas, 11 meningiomas, one pleomorphic xanthoastrocytoma, one angiomatous fibrous histiocytoma, 14 carcinoma metastases and seven gliomas grades II-IX) served as control. The pattern of strong, diffuse expression of carbonic anhydrase IX with membranous accentuation in combination with keratin negativity was considered diagnostic for hemangioblastomas. It was found in 18 out of 20 (90%) hemangioblastomas and in none of the control cases (P < 0.001). This resulted in a sensitivity of 90% and a specificity of 100%. The positive and negative predictive values were 100% and 96%, respectively. Carbonic anhydrase IX with cytokeratin is thus a highly sensitive and specific marker combination for hemangioblastomas. It is therefore very helpful in the diagnosis of these tumors and in their discrimination from other entities. PMID:25888144

  9. Characterization and inhibition studies of carbonic anhydrase from gill of Russian Sturgeon Fish (Acipenser gueldenstaedtii).

    PubMed

    Dinçer, Barbaros; Ekinci, Arife Pınar; Akyüz, Gülay; Kurtoğlu, İlker Zeki

    2016-12-01

    An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from gill of Acipenser gueldenstaedtii as an endangered sturgeon species. The carbonic anhydrase was purified 66-folds with yield 20.7% by Sepharose-4B-l-tyrosine-sulfanilamide affinity column and the specific activity was determined as 222.2 EU/mg protein. Km and Vmax kinetic values for gill carbonic anhydrase were calculated by a Lineweaver-Burk graph using p-nitrophenol acetate (p-NPA) as a substrate, and was defined as 2.5 mM and 5 × 10(6 )μM/min, respectively. It was observed that CA from the sturgeon gill in the presence of the sulfanilamide and acetazolamide as an inhibitor had very low IC50 values such as 13.0 and 0.1 μM, respectively. In addition, it was determined that the enzyme was inhibited by Fe(2+,) Co(2+,) Ni(2+), and Zn(2+)-Ba(2+) with the IC50 values of 0.2, 1.7, 1.2, and 1.1 mM, respectively.

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

    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.

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

    PubMed

    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

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

    PubMed Central

    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

  13. Carbonic anhydrase II is found in the placenta of a viviparous, matrotrophic lizard and likely facilitates embryo-maternal CO2 transport.

    PubMed

    Van Dyke, James U; Lindsay, Laura A; Murphy, Christopher R; Thompson, Michael B

    2015-11-01

    The evolution of viviparity requires the development of mechanisms that facilitate transport of respiratory gases between mother and developing embryo. Of particular importance is maternal excretion of embryonic carbon dioxide (CO2 ), which increases as the embryo grows in size during development. The carbonic anhydrases are a family of enzymes that convert CO2 to bicarbonate for transport throughout the cardiovascular system and which may also be important for CO2 transport from embryo to mother. We used immunohistochemistry to localize carbonic anhydrase II in the placental tissues of a viviparous and highly placentotrophic lizard, Pseudemoia entrecasteauxii. Carbonic anhydrase II is localized in the uterine component of the paraplacentome, presumably to facilitate transport of embryonic CO2 to the mother. Carbonic anhydrase II is also localized in both the uterine and embryonic components of the placentome, a region heavily involved in placental nutrient transport rather than respiratory gas exchange. In contrast, carbonic anhydrase II is not present in the uterine or embryonic components of the omphaloplacenta, another region responsible for nutrient transport. While carbonic anhydrase II in the paraplacentomal uterus is likely to be responsible for embryo-maternal CO2 transport, the distribution of carbonic anhydrase II throughout the placentome indicates a different function. Instead of transporting embryonic CO2 , placentomal carbonic anhydrase II appears to be responsible for transporting CO2 produced by energetically expensive nutrient transport mechanisms in both the uterus and the embryo, which implies that the mechanisms of nutrient transport in the omphaloplacenta may not be as energetically expensive.

  14. Cloning and expression of gamma carbonic anhydrase from Serratia sp. ISTD04 for sequestration of carbon dioxide and formation of calcite.

    PubMed

    Srivastava, Shaili; Bharti, Randhir Kumar; Verma, Praveen Kumar; Thakur, Indu Shekhar

    2015-01-01

    Bacterial strains isolated from marble mines rock and enriched in the chemostat culture with different concentrations of sodium bicarbonate. The enriched consortium had six bacterial isolates. One of bacterium isolate showed carbonic anhydrase (CA) activity by catalyzing the reversible hydration reaction of carbon dioxide to bicarbonate. The bacterium was identified as Serratia sp. by 16S rRNA sequence analysis. The carbonic anhydrase gene from Serratia sp. was found to be homologous with gamma carbonic anhydrase. The carbonic anhydrase gene was cloned in PET21b(+) and expressed it in recombinant Escherichia coli BL21 (DE3) with His-tag at the C-terminus. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. Expected size of carbonic anhydrase was approximately 29 kDa in SDS-PAGE gel. Recombinant carbonic anhydrase enzyme was used for biomineralization-based conversion of atmospheric CO2 into valuable calcite minerals. The calcification was confirmed by using XRD, FTIR, EDX and SEM analysis.

  15. Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

    PubMed Central

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

    2013-01-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

  16. Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

    PubMed Central

    Fisher, Zoë; Boone, Christopher D.; Biswas, Shya Masri; Venkatakrishnan, Balasubramanian; Aggarwal, Mayank; Tu, Chingkuang; Agbandje-McKenna, Mavis; Silverman, David; McKenna, Robert

    2012-01-01

    Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties. PMID:22691706

  17. Carbon Dioxide Sequestration by Using a Model Carbonic Anhydrase Complex in Tertiary Amine Medium.

    PubMed

    Sivanesan, Dharmalingam; Choi, Youngju; Lee, Jiyeon; Youn, Min Hye; Park, Ki Tae; Grace, Andrew Nirmala; Kim, Hak-Joo; Jeong, Soon Kwan

    2015-12-01

    Globally, the elevation of carbon dioxide (CO2 ) levels due to the anthropogenic effect poses a serious threat to the ecosystem. Hence, it is important to control and/or mitigate the level of CO2 in the atmosphere, which necessitates novel tools. Herein, it is proposed to improve CO2 sequestration by using model complexes based on the enzyme carbonic anhydrase (CA) in aqueous tertiary amine medium. The effect of substituents on the model CA model complexes on CO2 absorption and desorption was determined by using a stopped-flow spectrophotometer to follow pH changes through coupling to pH indicator and a continuous stirred-tank reactor (CSTR). The CO2 hydration rate constants were determined under basic conditions and compound 6, which contained a hydrophilic group, showed the highest absorption or hydration levels of CO2 (2.860×10(3)  L mol(-1)  s(-1) ). In addition, CSTR results for the absorption and desorption of CO2 suggest that simple model CA complexes could be used in post-combustion processing.

  18. Carbonic Anhydrase Inhibitors. Part 551 Metal Complexes of 1,3,4-Thiadiazole-2-Sulfonamide Derivatives: In Vitro Inhibition Studies With Carbonic Anhydrase Isozymes I, II and IV

    PubMed Central

    Scozzafava, Andrea; Briganti, Fabrizio; Ilies, Marc A.; Jitianu, Andrei

    1998-01-01

    Coordination compounds of 5-chloroacetamido-1,3,4-thiadiazole-2-sulfonamide (Hcaz) with V(IV), Cr(lll), Fe(ll), Co(ll), Ni(ll) and Cu(ll) have been prepared and characterized by standard procedures (spectroscopic, magnetic, EPR, thermogravimetric and conductimetric measurements). Some of these compounds showed very good in vitro inhibitory properties against three physiologically relevant carbonic anhydrase (CA)isozymes, i.e., CA I, II, and IV. The differences between these isozymes in susceptibility to inhibition by these metal complexes is discussed in relationship to the characteristic features of their active sites, and is rationalized in terms useful for developing isozyme-specific CA inhibitors. PMID:18475829

  19. Absence of carbonic anhydrase on the surface of red cells suspended in an elutriator

    SciTech Connect

    Dodek, P.; Effros, R.M.

    1986-03-01

    Studies of the carbonic anhydrase activity of intact red cells are complicated by hemolysis, which markedly accelerates catalytic conversion between HCO/sub 2//sup -/ and CO/sub 2/. The authors have reduced this effect to negligible levels by studying red cells suspended with an elutriator. 1.Oml of packed rabbit red cells were introduced in an elutriator chamber which was spun in a centrifuge with a force of 2900G. An isotonic, pH 7.4, 6/sup 0/C, HCO/sub 3//sup -/ Ringer's lactate solution was pumped centripetally through the chamber to keep the cells in suspension. 0.1 ml of the perfusion solution containing /sup 22/Na/sup +/, /sup 36/Cl-, H/sup 14/CO/sub 3//sup -///sup 14/CO/sub 2/ (equilibrated), and /sup 3/H/sub 2/O were injected into the elutriator inflow and 22 samples were collected with a computerized anaerobic collector. Outflow concentrations of each radionuclide were divided by the amounts injected to yield fractional concentrations and upslope areas of indicators were divided by that of /sup 22/Na/sup +/ to yield an index of red cell exchange, R. Under control conditions, R(/sup 14/C) averaged .80 +/- .01 (S.E.). After anion exchange had been blocked with 0.1..mu..M DIDS, R(/sup 14/C) increased to .86 +/- .01. When 100 mg/1 bovine carbonic anhydrase was added R(/sup 1/$C) fell to .78 +/- .01. Subsequent addition of acetazolamide (10/sup -5/) increased R(/sup 14/C) to .86 +/- .01. R(/sup 36/Cl/sup -/) was increased from .91 +/- .02 to .96 +/- .01 after DIDS. These data indicate no detectable carbonic anhydrase activity on red cell surface.

  20. A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

    PubMed Central

    Askoxylakis, Vasileios; Garcia-Boy, Regine; Rana, Shoaib; Krämer, Susanne; Hebling, Ulrike; Mier, Walter; Altmann, Annette; Markert, Annette; Debus, Jürgen; Haberkorn, Uwe

    2010-01-01

    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. PMID:21209841

  1. Carbonic Anhydrase Inhibition with Benzenesulfonamides and Tetrafluorobenzenesulfonamides Obtained via Click Chemistry

    PubMed Central

    2014-01-01

    A series of novel benzene- and 2,3,5,6-tetrafluorobenzenesulfonamide was synthesized by using a click chemistry approach starting from azido-substituted sulfonamides and alkynes, incorporating aryl, alkyl, cycloalkyl, and amino-/hydroxy-/halogenoalkyl moieties. The new compounds were medium potency inhibitors of the cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II and low nanomolar/subnanomolar inhibitors of the tumor-associated hCA IX and XII isoforms. The X-ray crystal structure of two such sulfonamides in adduct with hCA II allowed us to understand the factors governing inhibitory power. PMID:25147616

  2. Bidentate Zinc Chelators for α-Carbonic Anhydrases that Produce a Trigonal Bipyramidal Coordination Geometry

    PubMed Central

    Wischeler, Johannes Schulze; Innocenti, Alessio; Vullo, Daniela; Agrawal, Arpita; Cohen, Seth M.; Heine, Andreas; Supuran, Claudiu T.; Klebe, Gerhard

    2010-01-01

    A series of new zinc binding groups (ZBGs) has been evaluated kinetically on 13 carbonic anhydrase (CA) isoforms. The fragments show affinity for all isoforms with IC50 values in the range of 2–11 µm. The crystal structure of hCA II in complex with one such fragment reveals a bidentate binding mode with a trigonal-bipyramidal coordination geometry at the Zn2+ center. The fragment also interacts with Thr199 and Thr200 through hydrogen bonding and participates in a water network. Further development of this ZBG should increase the binding affinity leading to a structurally distinct and promising class of CA inhibitors. PMID:20629007

  3. Carbonic anhydrase inhibition by 1-aroyl-3-(4-aminosulfonylphenyl)thioureas.

    PubMed

    Saeed, Aamer; Al-Rashida, Mariya; Hamayoun, Mehwish; Mumtaz, Amara; Iqbal, Jamshed

    2014-12-01

    A series of 1-aroyl-3-(4-aminosulfonylphenyl)thioureas containing free sulfonamide group has been evaluated for their ability to inhibit bovine carbonic anhydrase II (bCA, EC 4.2.1.1). All compounds in the series were able to inhibit bCA II, the most active inhibitor had IC50 value of 0.26 ± 0.01 µM. Molecular docking studies and detailed structure-activity relationship studies were carried out. The absorption, distribution, metabolism, excretion (ADME) properties, as a predictor of oral absorption, were computationally calculated and compared with the clinically used drug acetazolamide. PMID:24666305

  4. Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis γ-carbonic anhydrase.

    PubMed

    Ceruso, Mariangela; Del Prete, Sonia; AlOthman, Zeid; Osman, Sameh M; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2014-08-15

    New benzenesulfonamides incorporating GABA or N-α-acetyl-L-lysine scaffolds as well as guanidine functionalities as water solubilizing moieties were obtained, using 4-aminoethyl/methyl-benzenesulfonamide and metanilamide/sulfanilamide as zinc-binding motives. The new compounds were medium potency inhibitors of the widespread cytosolic human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II and more effective inhibitors (KIs low nanomolar range) of the bacterial γ-CA from the oral pathogen Porphyromonas gingivalis. These sulfonamides may be useful tools for understanding the physiological role of bacterial CAs in pathogenesis of some infectious disease. PMID:25011913

  5. Synthesis and carbonic anhydrase inhibitory effects of new N-glycosylsulfonamides incorporating the phenol moiety.

    PubMed

    Riafrecha, Leonardo E; Bua, Silvia; Supuran, Claudiu T; Colinas, Pedro A

    2016-08-15

    A small series of N-glycosylsulfonamides incorporating the phenol moiety has been prepared by Ferrier sulfonamidoglycosylation of d-glycals. N-Glycosides were tested for the inhibition of four isoforms of carbonic anhydrase. In this study, all compounds showed good inhibitory activity against hCA I and II, with selectivity against the cytosolic hCA II versus the tumor associated isozymes. These results confirm that attaching carbohydrate moieties to CA phenol pharmacophore improves and enhances its inhibitory activity. PMID:27423482

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

    PubMed

    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

  7. New hydroxypyrimidinone-containing sulfonamides as carbonic anhydrase inhibitors also acting as MMP inhibitors.

    PubMed

    Esteves, M Alexandra; Ortet, Osvaldo; Capelo, Anabela; Supuran, Claudiu T; Marques, Sérgio M; Santos, M Amélia

    2010-06-15

    A set of benzenesulfonamide (BSA) derivatives bearing a hydroxypyrimidinone (HPM) moiety were synthesized and investigated for their inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes. They all revealed to be very potent inhibitors (nanomolar order) of the cytosolic CA I and II isozymes, but especially of the transmembrane, tumor-associated CA IX isozyme, a beneficial feature for a potential antitumor effect of these compounds. Further structure optimization aimed at improving the specificity of CA inhibition and enhancing their matrix metalloproteinase (MMP) inhibitory activity may also lead to new compounds with an attractive dual mechanism of action as antitumor agents.

  8. Pharmacotherapy of intraocular pressure - part II. Carbonic anhydrase inhibitors, prostaglandin analogues and prostamides.

    PubMed

    Costagliola, Ciro; dell'Omo, Roberto; Romano, Mario R; Rinaldi, Michele; Zeppa, Lucia; Parmeggiani, Francesco

    2009-12-01

    The second part of this two part review (please see Expert Opinion on Pharmacotherapy 10(16)) reports the characteristics of other antiglaucoma medications: systemic (acetazomide) and topical (dorzolamide and brinzolamide) carbonic anhydrase inhibitors, which suppress aqueous humour formation; and prostaglandin analogues (latanoprost and travoprost) and prostamides (bimatoprost), which raise aqueous humour outflow. The pharmacologic properties of each compound and its efficacy in the medical treatment of glaucoma, mainly the primary open-angle form, are discussed briefly, focusing on the clinical evidence supporting their use. PMID:19929706

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

    PubMed

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

    2012-02-11

    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 residues nearby. Several low nanomolar CA I, II and IX inhibitors were detected. PMID:22218610

  10. Saccharin sulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII.

    PubMed

    Morkūnaitė, Vaida; Baranauskienė, Lina; Zubrienė, Asta; Kairys, Visvaldas; Ivanova, Jekaterina; Trapencieris, Pēteris; Matulis, Daumantas

    2014-01-01

    A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1-10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1-10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

  11. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

    PubMed

    Mahon, Brian P; Hendon, Alex M; Driscoll, Jenna M; Rankin, Gregory M; Poulsen, Sally-Ann; Supuran, Claudiu T; McKenna, Robert

    2015-02-15

    Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

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

    PubMed Central

    Lionetto, Maria Giulia; Caricato, Roberto; Giordano, Maria Elena; Erroi, Elisa; Schettino, Trifone

    2012-01-01

    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. PMID:23202827

  13. Carbon dioxide capture using Escherichia coli expressing carbonic anhydrase in a foam bioreactor.

    PubMed

    Watson, Stuart K; Han, Zhenlin; Su, Wei Wen; Deshusses, Marc A; Kan, Eunsung

    2016-12-01

    The present study reports CO2 capture and conversion to bicarbonate using Escherichia coli expressing carbonic anhydrase (CA) on its cell surface in a novel foam bioreactor. The very large gas-liquid interfacial area in the foam bioreactor promoted rapid CO2 absorption while the CO2 in the aqueous phase was subsequently converted to bicarbonate ions by the CA. CO2 gas removal in air was investigated at various conditions such as gas velocity, cell density and CO2 inlet concentration. Regimes for kinetic and mass transfer limitations were defined. Very high removal rates of CO2 were observed: 9570 g CO2 m(-3) bioreactor h(-1) and a CO2 removal efficiency of 93% at 4% inlet CO2 when the gas retention time was 24 s, and cell concentration was 4 gdw L(-1). These performances are superior to earlier reports of experimental bioreactors using CA for CO2 capture. Overall, this bioreactor system has significant potential as an alternative CO2 capture technology.

  14. Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence.

    PubMed

    Bahn, Yong-Sun; Cox, Gary M; Perfect, John R; Heitman, Joseph

    2005-11-22

    The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen.

  15. CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

    PubMed

    Semenihin, A V; Zolotareva, O K

    2015-01-01

    Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase. PMID:26502699

  16. Structural study of X-ray induced activation of carbonic anhydrase

    PubMed Central

    Sjöblom, Björn; Polentarutti, Maurizio; Djinović-Carugo, Kristina

    2009-01-01

    Carbonic anhydrase, a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide to bicarbonate. It is involved in processes connected with acid–base homeostasis, respiration, and photosynthesis. More than 100 distinct human carbonic anhydrase II (HCAII) 3D structures have been generated in last 3 decades [Liljas A, et al. (1972) Nat New Biol 235:131–137], but a structure of an HCAII in complex with CO2 or HCO3− has remained elusive. Here, we report previously undescribed structures of HCAII:CO2 and HCAII:HCO3− complexes, together with a 3D molecular film of the enzymatic reaction observed successively in the same crystal after extended exposure to X-ray. We demonstrate that the unexpected enzyme activation was caused in an X-ray dose-dependent manner. Although X-ray damage to macromolecular samples has long been recognized [Ravelli RB, Garman EF (2006) Curr Opin Struct Biol 16:624–629], the detailed structural analysis reports on X-ray-driven reactions have been very rare in literature to date. Here, we report on enzyme activation and the associated chemical reaction in a crystal at 100 K. We propose mechanisms based on water photoradiolysis and/or electron radiolysis as the main cause of enzyme activation. PMID:19520834

  17. Structural study of X-ray induced activation of carbonic anhydrase.

    PubMed

    Sjöblom, Björn; Polentarutti, Maurizio; Djinovic-Carugo, Kristina

    2009-06-30

    Carbonic anhydrase, a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide to bicarbonate. It is involved in processes connected with acid-base homeostasis, respiration, and photosynthesis. More than 100 distinct human carbonic anhydrase II (HCAII) 3D structures have been generated in last 3 decades [Liljas A, et al. (1972) Nat New Biol 235:131-137], but a structure of an HCAII in complex with CO(2) or HCO(3)(-) has remained elusive. Here, we report previously undescribed structures of HCAII:CO(2) and HCAII:HCO(3)(-) complexes, together with a 3D molecular film of the enzymatic reaction observed successively in the same crystal after extended exposure to X-ray. We demonstrate that the unexpected enzyme activation was caused in an X-ray dose-dependent manner. Although X-ray damage to macromolecular samples has long been recognized [Ravelli RB, Garman EF (2006) Curr Opin Struct Biol 16:624-629], the detailed structural analysis reports on X-ray-driven reactions have been very rare in literature to date. Here, we report on enzyme activation and the associated chemical reaction in a crystal at 100 K. We propose mechanisms based on water photoradiolysis and/or electron radiolysis as the main cause of enzyme activation. PMID:19520834

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

    SciTech Connect

    Mitterberger, Maria C.; Kim, Geumsoo; Rostek, Ursula; Levine, Rodney L.; Zwerschke, Werner

    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.

  19. Kinetics of Formation of Cobalt(II)- and Nickel(II) Carbonic Anhydrase.

    ERIC Educational Resources Information Center

    McQuate, Robert S.; Reardon, John E.

    1978-01-01

    Discusses the kinetic behavior associated with the interaction of metal ions with apocarbonic anhydrase, focusing on the formation of two metallocarbonic anhydrase--the biochemically active Co(II) and the inactive Ni(II)derivatives. (GA)

  20. Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements.

    PubMed

    Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; Ries, Amber; Böhmer, Maik; You, Lei; Xiao, Chuanlei; Engineer, Cawas B; Boron, Walter F; Schroeder, Julian I

    2015-10-01

    Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3 (-) concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3 (-) concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified. PMID:26243620

  1. Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements1[OPEN

    PubMed Central

    Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; Ries, Amber; Böhmer, Maik; You, Lei; Xiao, Chuanlei; Engineer, Cawas B.; Boron, Walter F.; Schroeder, Julian I.

    2015-01-01

    Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3− concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3− concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified. PMID:26243620

  2. Intracellular and Extracellular Carbonic Anhydrases Cooperate Non-enzymatically to Enhance Activity of Monocarboxylate Transporters*

    PubMed Central

    Klier, Michael; Andes, Fabian T.; Deitmer, Joachim W.; Becker, Holger M.

    2014-01-01

    Proton-coupled monocarboxylate transporters (MCTs) are carriers of high-energy metabolites such as lactate, pyruvate, and ketone bodies and are expressed in most tissues. It has previously been shown that transport activity of MCT1 and MCT4 is enhanced by the cytosolic carbonic anhydrase II (CAII) independent of its catalytic activity. We have now studied the influence of the extracellular, membrane-bound CAIV on transport activity of MCT1/4, heterologously expressed in Xenopus oocytes. Coexpression of CAIV with MCT1 and MCT4 resulted in a significant increase in MCT transport activity, even in the nominal absence of CO2/HCO3−. CAIV-mediated augmentation of MCT activity was independent of the CAIV catalytic function, since application of the CA-inhibitor ethoxyzolamide or coexpression of the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT transport activity. The interaction required CAIV at the extracellular surface, since injection of CAIV protein into the oocyte cytosol did not augment MCT transport function. The effects of cytosolic CAII (injected as protein) and extracellular CAIV (expressed) on MCT transport activity, were additive. Our results suggest that intra- and extracellular carbonic anhydrases can work in concert to ensure rapid shuttling of metabolites across the cell membrane. PMID:24338019

  3. A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects.

    PubMed

    Carta, Fabrizio; Di Cesare Mannelli, Lorenzo; Pinard, Melissa; Ghelardini, Carla; Scozzafava, Andrea; McKenna, Robert; Supuran, Claudiu T

    2015-04-15

    A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known, and some appear to be involved in pain. These sulfonamides showed modest inhibition against the cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain relieving effect, a fact never observed earlier. This is the first report of rationally designed sulfonamide CA inhibitors with pain effective modulating effects. PMID:25766630

  4. The metanephros of the quail embryo. Developmental expression of carbonic anhydrase investigated by multiple approaches.

    PubMed

    Gabrielli, M G; Materazzi, G; Menghi, G

    2000-01-01

    The expression of carbonic anhydrase (CA) in the quail metanephros was investigated during embryonic development. The immunohistochemical localisation of the isoenzymes CAII and CAIII was compared with the distribution of enzyme activity visualised by a histochemical cobalt-precipitation procedure. The developmental profile of CA activity was also evaluated by means of a biochemical method. The occurrence of a moderate and diffuse CAII immunostaining from the first developmental appearance of the metanephros anlage testified to an early expression of carbonic anhydrase. This finding is discussed in relation to the involvement of the enzyme in the morphogenetic mechanisms leading to the establishment both of cell polarity and epithelial phenotype. CA expression in the renal sites that are positive in adults proved to be developmentally regulated. In the collecting duct system, enzyme activity could not be identified until the time of hatching. No CA was detected at any stage examined at the sites where, in adults, enzyme occurrence has previously been interpreted as a membrane-associated CA isoform. The differentiating renal tubules displayed no CAIII immunoreactivity. It can be argued that the bulk of the enzyme activity in the embryonic metanephros is due to the cytosolic isoenzyme CAII.

  5. Carbonic Anhydrase is Required for Statoconia Homeostasis in Organ Cultures of Statocysts from Aplysia californica

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

    A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (LI5) medium supplemented with salts and Aplysia haemolymph for four days at 17 C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(exp -4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially, via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.

  6. Structure of α-carbonic anhydrase from the human pathogen Helicobacter pylori.

    PubMed

    Compostella, Maria Elena; Berto, Paola; Vallese, Francesca; Zanotti, Giuseppe

    2015-08-01

    The crystal structure of α-carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacterium has corrected for the mutation of an essential amino acid important for catalysis using a negative ion from the medium and how it localizes close to the inner membrane in the periplasm. Since carbonic anhydrase is essential for the bacterial colonization of the host, it is a potential target for antibiotic drugs. The definition of the shape of the active-site entrance and cavity constitutes a basis for the design of specific inhibitors. PMID:26249690

  7. Catalase, carbonic anhydrase and xanthine oxidase activities in patients with mycosis fungoides.

    PubMed

    Cengiz, Fatma Pelin; Beyaztas, Serap; Gokce, Basak; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-04-01

    Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma. In several studies the relationship between catalase (CAT), human cytosolic carbonic anhydrases (CA; hCA-I and hCA-II) and xanthine oxidase (XO) enzyme activities have been investigated in various types of cancers but carbonic anhydrase, catalase and xanthine oxidase activities in patients with MF have not been previously reported. Therefore, in this preliminary study we aim to investigate CAT, CA and XO activities in patients with MF. This study enrolled 32 patients with MF and 26 healthy controls. According to the results, CA and CAT activities were significantly lower in patients with mycosis fungoides than controls (p < 0.001) (p < 0.001). There was no significant difference in XO activity between patient and control group (p = 0.601). Within these findings, we believe these enzyme activity levels might be a potentially important finding as an additional diagnostic biochemical tool for MF.

  8. Characteristics of recombinant α-carbonic anhydrase of polyextremophilic bacterium Bacillus halodurans TSLV1.

    PubMed

    Faridi, Shazia; Satyanarayana, T

    2016-08-01

    Carbonic anhydrase (CA) is a biocatalyst that catalyzes the hydration of CO2 to bicarbonate and protons, thus useful in mitigating green house effect by sequestering CO2 from various point sources. An alkalistable and moderately thermostable α- carbonic anhydrase encoding gene (BhCA) from Bacillus halodurans TSLV1 has been cloned and expressed in Escherichia coli. A 31.4-fold enhancement in CA production was achieved due to cloning and expression in E. coli. About 50% of the CA produced was secreted when recombinant E. coli with BhCA-pET22b was cultivated in a medium with EDTA and lysozyme because of the efficient pelB leader sequence. rBhCA is a ∼75kDa homodimeric protein with a Tm of 72°C and T1/2 values of 66 and 24min at 50 and 60°C, respectively. SDM analysis revealed that H137, H139, H156 and H110 present in the active site play an important role in catalysis. Mineralization of CO2 using rBhCA led to the accelerated precipitation of CaCO3 in calcite form. rBhCA also functions as an efficient virtual peroxidase when Zn(2+) is substituted with Mn(2+). PMID:27174908

  9. Exploring local flexibility/rigidity in psychrophilic and mesophilic carbonic anhydrases.

    PubMed

    Chiuri, R; Maiorano, G; Rizzello, A; del Mercato, L L; Cingolani, R; Rinaldi, R; Maffia, M; Pompa, P P

    2009-02-18

    Molecular flexibility and rigidity are required to determine the function and specificity of protein molecules. Some psychrophilic enzymes demonstrate a higher catalytic efficiency at low temperatures, compared to the efficiency demonstrated by their meso/thermophilic homologous. The emerging picture suggests that such enzymes have an improved flexibility of the structural catalytic components, whereas other protein regions far from functional sites may be even more rigid than those of their mesophilic counterparts. To gain a deeper insight in the analysis of the activity-flexibility/rigidity relationship in protein structure, psychrophilic carbonic anhydrase of the Antarctic teleost Chionodraco hamatus has been compared with carbonic anhydrase II of Bos taurus through fluorescence studies, three-dimensional modeling, and activity analyses. Data demonstrated that the cold-adapted enzyme exhibits an increased catalytic efficiency at low and moderate temperatures and, more interestingly, a local flexibility in the region that controls the correct folding of the catalytic architecture, as well as a rigidity in the hydrophobic core. The opposite result was observed in the mesophilic counterpart. These results suggest a clear relationship between the activity and the presence of flexible and rigid protein substructures that may be useful in rational molecular and drug design of a class of enzymes playing a key role in pathologic processes.

  10. Carbonic anhydrase, a respiratory enzyme in the gills of the shore crab Carcinus maenas

    NASA Astrophysics Data System (ADS)

    Böttcher, K.; Siebers, D.; Sender, S.

    1995-03-01

    This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crab Carcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO2 to HCO3 - and H+, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crab Callinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO3 - and H+ for apical exchange against Na+ and Cl-, the involvement of CA in NaCl uptake mechanisms can be excluded in Carcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) Ki=2.4·10-8 mol/l). Using the preparation of the isolated perfused gill, application of 10-4 mol/l AZ resulted in an 80% decrease of CO2/HCO3 - excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO2 gradient by accelerating adjustment of the pH-dependent CO2/HCO3 - equilibrium.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog

    PubMed Central

    Premkumar, Lakshmanane; Greenblatt, Harry M.; Bageshwar, Umesh K.; Savchenko, Tatyana; Gokhman, Irena; Sussman, Joel L.; Zamir, Ada

    2005-01-01

    Protein molecular adaptation to drastically shifting salinities was studied in dCA II, an α-type carbonic anhydrase (EC 4.2.1.1) from the exceptionally salt-tolerant unicellular green alga Dunaliella salina. The salt-inducible, extracellular dCA II is highly salt-tolerant and thus differs from its mesophilic homologs. The crystal structure of dCA II, determined at 1.86-Å resolution, is globally similar to other α-type carbonic anhydrases except for two extended α-helices and an added Na-binding loop. Its unusual electrostatic properties include a uniformly negative surface electrostatic potential of lower magnitude than that observed in the highly acidic halophilic proteins and an exceptionally low positive potential at a site adjoining the catalytic Zn2+ compared with mesophilic homologs. The halotolerant dCA II also differs from typical halophilic proteins in retaining conformational stability and solubility in low to high salt concentrations. The crucial role of electrostatic features in dCA II halotolerance is strongly supported by the ability to predict the unanticipated halotolerance of the murine CA XIV isozyme, which was confirmed biochemically. A proposal for the functional significance of the halotolerance of CA XIV in the kidney is presented. PMID:15894606

  13. Identification, sequencing, and localization of a new carbonic anhydrase transcript from the hydrothermal vent tubeworm Riftia pachyptila.

    PubMed

    Sanchez, Sophie; Andersen, Ann C; Hourdez, Stéphane; Lallier, François H

    2007-10-01

    The vestimentiferan annelid Riftia pachyptila forms dense populations at hydrothermal vents along the East Pacific Rise at a depth of 2600 m. It harbors CO(2)-assimilating sulfide-oxidizing bacteria that provide all of its nutrition. To find specific host transcripts that could be important for the functioning of this symbiosis, we used a subtractive suppression hybridization approach to identify plume- or trophosome-specific proteins. We demonstrated the existence of carbonic anhydrase transcripts, a protein endowed with an essential role in generating the influx of CO(2) required by the symbionts. One of the transcripts was previously known and sequenced. Our quantification analyses showed a higher expression of this transcript in the trophosome compared to the branchial plume or the body wall. A second transcript, with 69.7% nucleotide identity compared to the previous one, was almost only expressed in the branchial plume. Fluorescent in situ hybridization confirmed the coexpression of the two transcripts in the branchial plume in contrast with the trophosome where only one transcript could be detected. An alignment of these translated carbonic anhydrase cDNAs with vertebrate and nonvertebrate carbonic anhydrase protein sequences revealed the conservation of most amino acids involved in the catalytic site. According to the phylogenetic analyses, the two R. pachyptila transcripts clustered together but not all nonvertebrate sequences grouped together. Complete sequencing of the new carbonic anhydrase transcript revealed the existence of two slightly divergent isoforms probably coded by two different genes. PMID:17892492

  14. Crystallization and preliminary X-ray diffraction studies of a beta-carbonic anhydrase from the red alga Porphyridium purpureum.

    PubMed

    Mitsuhashi, S; Mizushima, T; Yamashita, E; Miyachi, S; Tsukihara, T

    2000-02-01

    The beta-carbonic anhydrase from the red alga Porphyridium purpureum was heterologously expressed, purified and crystallized. The crystals belong to space group P2(1) (unit-cell parameters a = 63.8, b = 113.9, c = 73.8 A, beta = 104.1 degrees) with two subunits per asymmetric unit and diffract to 2.5 A resolution.

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

    PubMed Central

    Belkaid, Anissa; Čuperlović-Culf, Miroslava; Touaibia, Mohamed; Ouellette, Rodney J.; Surette, Marc E.

    2016-01-01

    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. PMID:27240414

  16. Acute effects of thiazides, with and without carbonic anhydrase inhibiting activity, on lithium and free water clearance in man.

    PubMed

    Boer, W H; Koomans, H A; Dorhout Mees, E J

    1989-05-01

    1. The acute effects of chlorothiazide and bendroflumethiazide on renal Li+ clearance (CLi) were studied in Na+-restricted healthy humans during maximum water diuresis. 2. Chlorothiazide, which has marked carbonic anhydrase inhibiting activity, increased CLi by about 25%. The concomitant rise in uric acid clearance, maximum urine flow and bicarbonate excretion suggests that this drug suppressed proximal reabsorption through carbonic anhydrase inhibition, which would also explain the observed fall in glomerular filtration rate (increased glomerulotubular feedback activity). 3. Bendroflumethiazide, which lacks carbonic anhydrase inhibiting activity, did not affect CLi or any of the other above-mentioned variables. 4. It is concluded from the lack of an effect of bendroflumethiazide on CLi that Li+ is not reabsorbed in thiazide-sensitive segments of the human distal nephron. The rise in CLi after chlorothiazide is most likely due to suppressed Li+ reabsorption in the proximal tubules resulting from carbonic anhydrase inhibition. 5. The results of this study are compatible with the concept that CLi is an index of Na+ and water delivery from the proximal tubules in humans. PMID:2721120

  17. Role of carbonic anhydrases in pathogenic micro-organisms: a focus on Aspergillus fumigatus.

    PubMed

    Tobal, Jaqueline Moisés; Balieiro, Márcia Eliana da Silva Ferreira

    2014-01-01

    Aspergillus fumigatus is a ubiquitous saprophytic fungus responsible for organic material decomposition, and plays an important role in recycling environmental carbon and nitrogen. Besides its important role in the environment, this fungus has been reported as one of the most important fungal pathogens in immunocompromised patients. Due to changes in CO2 concentration that some pathogens face during the infection process, studies have been undertaken to understand the pathogenic roles of carbonic anhydrases (CAs), well-known CO2 hydration catalytic enzymes. As a basis for a discussion of the possible roles of CAs in A. fumigatus pathogenicity, this review describes the main characteristics of the A. fumigatus infection and the challenges for its treatment. In addition, it gathers findings from studies with CA inhibitor drugs as anti-infective agents in different pathogens.

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

    PubMed

    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

  19. Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo.

    PubMed

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

    2012-02-23

    A series of dithiocarbamates were prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four 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. The X-ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an effective intraocular pressure lowering activity in an animal model of glucoma. PMID:22276570

  20. Macromolecular conformation in solution. Study of carbonic anhydrase by the positron annihilation technique.

    PubMed Central

    Handel, E D; Graf, G; Glass, J C

    1980-01-01

    The structural features of carbonic anhydrase (carbonate hydro-lyase; EC 4.2.1.1) in aqueous solution were probed by the positron annihilation technique. The data obtained under varying conditions of temperature, pH, and enzyme concentration were interpreted in terms of the free volume model. The change of enzymic activity with temperature is accompanied by a change in free volume of the protein. Upon thermal denaturation an irreversible change in free volume of the molecule occurred. At low temperatures the protein-water interactions were investigated. These results are discussed in terms of current concepts of structure-function relationships in proteins. This study shows the sensitivity of the positron annihilation method toward the structure of proteins related to their overall conformation and to the nature of bound water. PMID:6789901

  1. [Advances in researches on β-carbonic anhydrases as anti-parasitic drug targets].

    PubMed

    Zhang, Cong-hui; Zhu, Huai-min

    2016-02-01

    β-carbonic anhydrases (β-CAs) are ubiquitous metalloenzymes which active site contains a zinc ion (Zn²⁺), and they could catalyze the hydration of carbon dioxide to bicarbonate and protons efficiently and are involved in many biological processes, such as respiration, pH and CO₂ homeostasis, biosynthetic reactions, virulence regulation and so on, and may play a critical role in the life activity of many organisms which contain these enzymes. β-CAs are widely distributed in fungi, bacteria, algae, plants and a small number of protozoan and metazoan except vertebrates. Therefore, as potential drug targets for designing and developing antibacterial and anti-parasitic drugs, β-CAs promise a broad application prospect. This paper focuses on the distribution, physiological function and the progress of researches on β-CAs in parasites and their vectors. PMID:27356420

  2. Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo#

    PubMed Central

    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 evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed effective intraocular pressure lowering activity in an animal model of glucoma. PMID:22276570

  3. Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

    NASA Astrophysics Data System (ADS)

    Engineer, Cawas B.; Ghassemian, Majid; Anderson, Jeffrey C.; Peck, Scott C.; Hu, Honghong; Schroeder, Julian I.

    2014-09-01

    Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding of

  4. Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development.

    PubMed

    Engineer, Cawas B; Ghassemian, Majid; Anderson, Jeffrey C; Peck, Scott C; Hu, Honghong; Schroeder, Julian I

    2014-09-11

    Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding

  5. Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active beta-carbonic anhydrase from Mycobacterium tuberculosis, Rv3588c.

    PubMed

    Carta, Fabrizio; Maresca, Alfonso; Covarrubias, Adrian Suarez; Mowbray, Sherry L; Jones, T Alwyn; Supuran, Claudiu T

    2009-12-01

    The Rv3588c gene product of Mycobacterium tuberculosis, a beta-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO(2) hydration (k(cat) of 9.8 x 10(5)s(-1), and k(cat)/K(m) of 9.3 x 10(7)M(-1)s(-1)) among the three beta-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K(I)s of 9-59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action. PMID:19846301

  6. Carbonic anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic halves of the carbonic anhydrase active site.

    PubMed

    Tanpure, Rajendra P; Ren, Bin; Peat, Thomas S; Bornaghi, Laurent F; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2015-02-12

    We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

  7. Carbonic Anhydrases in Cnidarians: Novel Perspectives from the Octocorallian Corallium rubrum

    PubMed Central

    Le Goff, Carine; Ganot, Philippe; Zoccola, Didier; Caminiti-Segonds, Natacha; Allemand, Denis; Tambutté, Sylvie

    2016-01-01

    Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs. PMID:27513959

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

    SciTech Connect

    Budayova-Spano, Monika; Fisher, S. Zoë; Dauvergne, Marie-Thérèse; Agbandje-McKenna, Mavis; Silverman, David N.; Myles, Dean A. A.; McKenna, Robert

    2006-01-01

    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 (H{sub 2}O/OH{sup −}) 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.

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

    PubMed

    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.

  10. Carbonic anhydrase: a key regulatory and detoxifying enzyme for Karst plants.

    PubMed

    Müller, Werner E G; Qiang, Li; Schröder, Heinz C; Hönig, Natalie; Yuan, Daoxian; Grebenjuk, Vlad A; Mussino, Francesca; Giovine, Marco; Wang, Xiaohong

    2014-01-01

    Karstification is a rapid process during which calcidic stones/limestones undergo dissolution with the consequence of a desertification of karst regions. A slow-down of those dissolution processes of Ca-carbonate can be approached by a reforestation program using karst-resistant plants that can resist alkaline pH and higher bicarbonate (HCO₃⁻) concentrations in the soil. Carbonic anhydrases (CA) are enzymes that mediate a rapid and reversible interconversion of CO₂ and HCO₃⁻. In the present study, the steady-state expression of a CA gene, encoding for the plant carbonic anhydrase from the parsley Petroselinum crispum, is monitored. The studies were primarily been performed during germination of the seeds up to the 12/14-day-old embryos. The CA cDNA was cloned. Quantitative polymerase chain reaction (qPCR) analysis revealed that the gene expression level of the P. crispum CA is strongly and significantly affected at more alkaline pH in the growth medium (pH 8.3). This abolishing effect is counteracted both by addition of HCO₃⁻ and by addition of polyphosphate (polyP) to the culture medium. In response to polyP, the increased pH in the vacuoles of the growing plants is normalized. The effect of polyP let us to propose that this polymer acts as a buffer system that facilitates the adjustment of the pH in the cytoplasm. In addition, it is proposed that polyP has the potential to act, especially in the karst, as a fertilizer that allows the karstic plants to cope with the adverse pH and HCO₃⁻ condition in the soil. PMID:24385198

  11. Carbonic Anhydrases in Cnidarians: Novel Perspectives from the Octocorallian Corallium rubrum.

    PubMed

    Le Goff, Carine; Ganot, Philippe; Zoccola, Didier; Caminiti-Segonds, Natacha; Allemand, Denis; Tambutté, Sylvie

    2016-01-01

    Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs. PMID:27513959

  12. Silencing of carbonic anhydrase in an Anopheles gambiae larval cell line, Ag55.

    PubMed

    Smith, Kristin E; Linser, Paul J

    2009-06-17

    RNAi has been used extensively to down-regulate proteins in adult mosquitoes; however, it is not well adapted for use in larvae. Larval mosquitoes can generate a pH as high as 10.5 in the anterior region of their midgut. The mechanisms responsible for the generation and maintenance of this pH are not entirely understood, but members of the carbonic anhydrase (CA) family of enzymes have been implicated. Here we use an An. gambiae larval cell line, Ag55 cells, to demonstrate that application of full-length double-stranded RNA specific to one CA, AgCA9, is sufficient to silence AgCA9 mRNA and down-regulate the corresponding protein. This is a first step towards determining the role(s) of these enzymes in pH regulation.

  13. Lactate flux in astrocytes is enhanced by a non-catalytic action of carbonic anhydrase II

    PubMed Central

    Stridh, Malin H; Alt, Marco D; Wittmann, Sarah; Heidtmann, Hella; Aggarwal, Mayank; Riederer, Brigitte; Seidler, Ursula; Wennemuth, Gunther; McKenna, Robert; Deitmer, Joachim W; Becker, Holger M

    2012-01-01

    Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a ‘proton collecting antenna’ for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate. PMID:22451434

  14. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

    PubMed

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

    2015-09-01

    The Antarctic bacterium Pseudoalteromonas haloplanktis encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (PhaCAγ) has a good catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) of 1.4×10(5) s(-1) and a k(cat)/K(m) of 1.9×10(6) M(-1)×s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. Methazolamide and indisulam showed the best inhibitory properties (K(I)s of 86.7-94.7 nM). This contribution shed new light on γ-CAs inhibition profiles with a relevant class of pharmacologic agents.

  15. Carbonic Anhydrase Inhibitors: Design, Synthesis, and Biological Evaluation of Novel Sulfonyl Semicarbazide Derivatives

    PubMed Central

    2014-01-01

    A series of novel sulfonyl semicarbazides 5–13 was designed, synthesized, and evaluated for human carbonic anhydrase (hCA) inhibition. The new sulfonyl semicarbazides were tested against a panel of hCA isoforms I, II, IX, and XII, using acetazolamide (AZA, 1) as standard. All the sulfonyl semicarbazides showed subnanomolar affinity for hCA XII (pKi range 0.59–0.79 nM) and high selectivity over hCA I (58–114-fold) and hCA IX (26–114-fold) compared to hCA II (5–20-fold except 11, 121-fold). The importance of the nature of para-substitution on the sulfonyl substituted aromatic ring for potency and selectivity against one hCA isoform versus others is discussed. Overall, the research work led to the development of highly potent and selective hCA inhibitors. PMID:25050167

  16. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with Schiff's bases incorporating iminoureido moieties.

    PubMed

    Singasane, Namrata; Kharkar, Prashant S; Ceruso, Mariangela; Supuran, Claudiu T; Toraskar, Mrunmayee P

    2015-12-01

    A series of new Schiff's bases was obtained from the sulfanilamide semicarbazone (4-aminosulfonylphenyl semicarbazide) and aromatic/heterocyclic aldehydes. The new compounds were designed to incorporate moieties known to induce effective inhibitory activity against carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in crucial physiologic or pathologic processes such as the cytosolic CA I and II or the transmembrane, tumor-associated CA IX and XII: the compounds were medium potency - weak CA I inhibitors, highly effective, low nanomolar CA II inhibitors, but few of them inhibited effectively CA IX and XII. This may probably due to the long spacer between the sulfamoylphenyl and imine fragments of the molecules, which probably induces a highly flexible conformation of the inhibitor bound to the active site of the enzyme, with destabilizing effects for the adduct. The detailed structure activity relationship for this class of inhibitors is discussed. PMID:25744513

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

    PubMed

    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. Synthesis and evaluation of N-heteroarylsubstituted triazolosulfonamides as carbonic anhydrase inhibitors.

    PubMed

    Balci, Ahmet; Arslan, Mustafa; Nixha, Arleta Rifati; Bilen, Cigdem; Ergun, Adem; Gençer, Nahit

    2015-06-01

    A new series of N-heteroarylsubstituted triazolosulfonamide compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and II were evaluated. Compounds (3 a-k) were prepared by propargylation of N-heteroaryl compounds. Compound 5 was obtained from sulfanilamide and sodium nitrite followed by addition of sodium azide. The products (6 a-k) were synthesized from compounds 3 and 5. The results showed that all the synthesized compounds were inhibited the CA isoenzymes activity. Figure 6a (IC50 = 0.52 µM for hCA I and 0.34 µM for hCA II) has the most inhibitory effect among the synthesized compounds. PMID:25068730

  19. Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

    PubMed Central

    Warden, Andrew C.; Williams, Michelle; Peat, Thomas S.; Seabrook, Shane A.; Newman, Janet; Dojchinov, Greg; Haritos, Victoria S.

    2015-01-01

    Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na+ hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated. PMID:26687908

  20. Synthesis and Biological Evaluation of Novel Bischalcone Derivatives as Carbonic Anhydrase Inhibitors.

    PubMed

    Arslan, Tayfun; Çelik, Gonca; Çelik, Habip; Şentürk, Murat; Yaylı, Nurettin; Ekinci, Deniz

    2016-09-01

    Design and synthesis of a new type of bischalcones as an alternative to natural and synthetic bischalcones are reported for the first time. Key steps involved the solvent-free Claisen-Schmidt condensation of chalcones, and the successful first application of the diazotization-diazocoupling reaction in the synthesis of CNNC-linked bischalcones by simple structural modification of p-aminoacetophenone. The structures of all compounds were confirmed by means of FT-IR, (1) H and (13) C NMR, ESI/MS, and elemental analysis. In addition, the newly synthesized compounds were screened for carbonic anhydrase inhibition activities. Almost all bischalcones exhibited moderate-to-good inhibitory activities. PMID:27435458

  1. Carbonic Anhydrase Inhibitors. Part 541: Metal Complexes of Heterocyclic Sulfonamides: A New Class of Antiglaucoma Agents

    PubMed Central

    Scozzafava, Andrea; Jitianu, Andrei

    1997-01-01

    Metal complexes of heterocyclic sulfonamides possessing carbonic anhydrase (CA) inhibitory properties were recently shown to be useful as intraocular pressure (IOP) lowering agents in experimental animals, and might be developed as a novel class of antiglaucoma drugs. Here we report the synthesis of a heterocyclic sulfonamide CA inhibitor and of the metal complexes containing main group metal ions, such as Be(II), Mg(II), Al(III), Zn(II), Cd(II) and Hg(II) and the new sulfonamide as well as 5-amino-1,3,4-thiadiazole-2-sulfonamide as ligands. The new complexes were characterized by standard physico-chemical procedures, and assayed as inhibitors of three CA isozymes, CA I, II and IV. Some of them (but not the parent sulfonamides) strongly lowered IOP in rabbits when administered as a 2% solution into the eye. PMID:18475811

  2. Increased levels of carbonic anhydrase II in the developing Down syndrome brain.

    PubMed

    Palminiello, Sonia; Kida, Elizabeth; Kaur, Kulbir; Walus, Marius; Wisniewski, Krystyna E; Wierzba-Bobrowicz, Teresa; Rabe, Ausma; Albertini, Giorgio; Golabek, Adam A

    2008-01-23

    By using a proteomic approach, we found increased levels of carbonic anhydrase II (CA II) in the brain of Ts65Dn mice, a mouse model for Down syndrome (DS). Further immunoblot analyses showed that the levels of CA II are increased not only in the brain of adult Ts65Dn mice but also in the brain of infants and young children with DS. Cellular localization of the enzyme in human brain, predominantly in the oligodendroglia and primitive vessels in fetal brain and in the oligodendroglia and some GABAergic neurons postnatally, was similar in DS subjects and controls. Given the role of CA II in regulation of electrolyte and water balance and pH homeostasis, up-regulation of CA II may reflect a compensatory mechanism mobilized in response to structural/functional abnormalities in the developing DS brain. However, this up-regulation may also have an unfavorable effect by increasing susceptibility to seizures of children with DS.

  3. Tyrosine nitration provokes inhibition of sunflower carbonic anhydrase (β-CA) activity under high temperature stress.

    PubMed

    Chaki, Mounira; Carreras, Alfonso; López-Jaramillo, Javier; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Valderrama, Raquel; Corpas, Francisco J; Barroso, Juan B

    2013-02-28

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) and it is a new area of research in higher plants. Previously, it was demonstrated that the exposition of sunflower (Helianthus annuus L.) seedlings to high temperature (HT) caused both oxidative and nitrosative stress. The nitroproteome analysis under this stress condition showed the induction of 13 tyrosine-nitrated proteins being the carbonic anhydrase (CA) one of these proteins. The analysis of CA activity under high temperature showed that this stress inhibited the CA activity by a 43%. To evaluate the effect of nitration on the CA activity in sunflower it was used 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent. Thus the CA activity was inhibited by 41%. In silico analysis of the pea CA protein sequence suggests that Tyr(205) is the most likely potential target for nitration.

  4. Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

    PubMed Central

    Pinard, Melissa A.

    2015-01-01

    The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3−. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors. PMID:25811028

  5. Dithiocarbamates strongly inhibit the β-class carbonic anhydrases from Mycobacterium tuberculosis.

    PubMed

    Maresca, Alfonso; Carta, Fabrizio; Vullo, Daniela; Supuran, Claudiu T

    2013-04-01

    A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA 1 (Rv1284) and mtCA 3 (Rv3273). Both enzymes were inhibited with efficacies between the subnanomolar to the micromolar one, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. Aryl, arylalkyl-, heterocyclic as well as aliphatic and amino acyl such moieties led to potent mtCA 1 and 3 inhibitors in both the N-mono- and N,N-disubstituted dithiocarbamate series. This new class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance. PMID:22145736

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

    PubMed Central

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

    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 new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences. PMID:26797606

  7. Honey, pollen, and propolis extracts show potent inhibitory activity against the zinc metalloenzyme carbonic anhydrase.

    PubMed

    Sahin, H; Aliyazicioglu, R; Yildiz, O; Kolayli, S; Innocenti, A; Supuran, C T

    2011-06-01

    Three different honey extracts from the endemic plant in the Black Sea region Rhododendron ponticum, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), more precisely the human (h) isoforms hCA I and hCA II. Hexane, methanol, ethanol, and water solid-phase extractions (SPEs) showed inhibitory activity towards the two CA isozymes which were related to the total phenolic content. The highest inhibitory effects (0.036-0.039 mg/mL) were those of propolis methanolic extract. Among the three different samples investigated here, the aqueous extracts showed lower inhibitory effects compared to the organic solvent SPE extracts (in the range of 1.150- 5.144 mg/mL). The studied honey extracts constitute an interesting source of phenolic derivatives that might serve to identify lead compounds, targeting the physiologically relevant enzymes CA I and CA II.

  8. Expression of a carbonic anhydrase gene is induced by environmental stresses in rice (Oryza sativa L.).

    PubMed

    Yu, Song; Zhang, Xinxin; Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

    2007-01-01

    Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO(3) and Na(2)CO(3)), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO(3), 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice. PMID:17016673

  9. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

    PubMed

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

    2015-09-01

    The Antarctic bacterium Pseudoalteromonas haloplanktis encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (PhaCAγ) has a good catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) of 1.4×10(5) s(-1) and a k(cat)/K(m) of 1.9×10(6) M(-1)×s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. Methazolamide and indisulam showed the best inhibitory properties (K(I)s of 86.7-94.7 nM). This contribution shed new light on γ-CAs inhibition profiles with a relevant class of pharmacologic agents. PMID:26174556

  10. Pyridinium derivatives of histamine are potent activators of cytosolic carbonic anhydrase isoforms I, II and VII.

    PubMed

    Dave, Khyati; Scozzafava, Andrea; Vullo, Daniela; Supuran, Claudiu T; Ilies, Marc A

    2011-04-21

    A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested--valuable lead compounds for physiology and pathology studies involving these isozymes.

  11. Brain but not retinal glial cells have carbonic anhydrase activity in the honeybee drone.

    PubMed

    Walz, B

    1988-02-15

    Carbonic anhydrase (CA) activity was localized histochemically in the retina and brain of the honeybee drone. A positive reaction that could be inhibited with 10(-5) M acetazolamide was found only in brain glial cells such as those in the lamina and medulla of the optic lobes. In the retina, neither the photoreceptors nor the pigmented glial cells showed CA activity. Hence, there is a marked difference between retinal and brain glial cells with respect to those functions thought to be performed by CA. This study extends the range of tissues in which CA has been shown to be localized in glial cells, but the absence of CA from the retina will impose constraints on a general explanation of the role of CA in nervous tissue.

  12. Effect of Carbonic Anhydrase II in Molten Globule State on Physical Properties of Dimyristoylphosphatidylcholine Liposome

    NASA Astrophysics Data System (ADS)

    Sakai, Hiroko; Tanaka, Michiko; Imai, Kenichiro; Sonoyama, Masashi; Mitaku, Shigeki

    2001-05-01

    Ultrasonic relaxation measurement was employed for confirmation of the interaction between dimyristoylphosphatidylcholine (DMPC) membrane and a soluble protein, carbonic anhydrase II (CA II). The enhancement of the fluctuation of DMPC membrane structure was observed in the presence of CA II under acidic condition, pH 3.6-4, indicating the interaction between DMPC and CA II@. The pyrene fluorescence spectrum of CA II solution clearly showed that this protein adopted an unfolding intermediate called the molten globule state under the low pH condition, in which CA II interacted with DMPC@. However, CA II in the molten globule state did not cause membrane lysis in contrast to the high lytic activity of α-lactalbumin on DMPC liposomes.

  13. Carbonic anhydrase-facilitated CO2 absorption with polyacrylamide buffering bead capture

    SciTech Connect

    Dilmore, Robert; Griffith, Craid; Liu, Zhu; Soong, Yee; Hedges, Sheila W.; Koepsel, Richard; Ataai, M

    2009-07-01

    A novel CO2 separation concept is described wherein the enzyme carbonic anhydrase (CA) is used to increase the overall rate Of CO2 absorption after which hydrated CO2 reacts with regenerable amine-bearing polyacrylamide buffering beads (PABB). Following saturation of the material's immobilized tertiary amines, CA-bearing carrier water is separated and recycled to the absorption stage while CO2-loaded material is thermally regenerated. Process application of this concept would involve operation of two or more columns in parallel with thermal regeneration with low-pressure steam taking place after the capacity of a column of amine-bearing polymeric material was exceeded. PABB CO2- bearing capacity was evaluated by thermogravimetric analysis (TGA) for beads of three acrylamido buffering monomer ingredient concentrations: 0 mol/kg bead, 0.857 mol/kg bead, and 2 mol/kg bead. TGA results demonstrate that CO2- bearing capacity increases with increasing PABB buffering concentration and that up to 78% of the theoretical CO2- bearing capacity was realized in prepared PABB samples (0.857 mol/kg recipe). The highest observed CO2-bearing capacity of PABB was 1.37 mol of CO2 per kg dry bead. TGA was also used to assess the regenerability Of CO2-loaded PABB. Preliminary results suggest that CO2 is partially driven from PABB samples at temperatures as low as 55 degrees C, with complete regeneration occurring at 100 degrees C. Other physical characteristics of PABB are discussed. In addition, the effectiveness of bovine carbonic anhydrase for the catalysis Of CO2 dissolution is evaluated. Potential benefits and drawbacks of the proposed process are discussed. Published by Elsevier Ltd.

  14. Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II

    PubMed Central

    Nettles, Whitnee L.; Song, He; Farquhar, Erik R.; Fitzkee, Nicholas C.; Emerson, Joseph P.

    2015-01-01

    Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo-form of human carbonic anhydrase II (CA) binds two equivalents of copper(II) with high affinity. The Cu2+ ions bind independently forming two non-coupled type-II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we have identified an N-terminal Cu2+ binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four to five coordinate N-terminal Cu2+ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-ATCUN and copper(II)-beta-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu2+ binding data to the high-affinity CuA site, we have derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu2+ binding to the CuB site of CA to be 2 × 109 and −17.4 kcal/mol, respectively. PMID:26010488

  15. Carbonic anhydrase and acid base balance in relation to thermal stress in buffaloes ( Bubalus bubalis)

    NASA Astrophysics Data System (ADS)

    Mehta, S. N.; Gangwar, P. C.

    1983-03-01

    The blood samples from fifteen normal lactating buffaloes were taken from December 15th 1978 to 31st August, 1979. Depending upon the climatic conditions, the whole period of study was divided into four seasons. The mean values of carbonic anhydrase (moles CO2/l/sec×10-5) were 3.08±0.26, 4.94±0.44, 5.23±0.35, 6.44±0.32 in pregnant and 4.87±0.27, 4.53±0.41, 4.74±0.45, 6.36±0.40 in non-pregnant animals during winter, spring, hot and dry and hot and humid seasons. Mean values of pO2 (mm Hg) were 31.26±1.41, 31.92±0.61, 35.90±0.59, 33.80 ±0.67 in pregnant and 31.89±0.44, 31.53±0.54, 35.52±0.69, 31.65±0.95 in non-pregnant buffaloes during winter, spring, hot and dry and hot and humid periods, respectively. There were highly significant (P< 0.01) differences between seasons with respect to pO2, pCO2, actual HCO3 and heamoglobin. However, PCV changed significantly (P<0.01) with the physiological status of the animal. Different correlation of biochemical parameters with climatic elements were discussed. Thus, the shifts in the levels of carbonic anhydrase, HCO3 and heamoglobin may prove to be a better tool/index for thermal stress in buffaloes.

  16. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    SciTech Connect

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. )

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  17. Isolation and characterization of 2-nitroimidazole produced by Streptomyces species as an inhibitor of both carbonic anhydrase and shell formation in the barnacle Balanus amphitrite.

    PubMed

    Fukushima, Mari; Ozaki, Noriaki; Ikeda, Hiroyuki; Furihata, Keiko; Hayakawa, Yoichi; Sakuda, Shohei; Nagasawa, Hiromichi

    2002-03-01

    Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.

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

    PubMed Central

    2010-01-01

    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. PMID:20659325

  19. Immobilization of carbonic anhydrase enzyme purified from Bacillus subtilis VSG-4 and its application as CO(2) sequesterer.

    PubMed

    Oviya, M; Giri, Sib Sankar; Sukumaran, V; Natarajan, P

    2012-01-01

    The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan-alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co(2+), Cu(2+), and Fe(3+), increased the enzyme activity, whereas CA activity was inhibited by Pb(2+), Hg(2+), ethylenediamine tetraacetic acid (EDTA), 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO(2) to CaCO(3). The maximum CO(2) sequestration potential was achieved with immobilized CA (480 mg CaCO(3)/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO(2) sequestration. PMID:22897768

  20. The role of carbonic anhydrase in C4 photosynthesis

    SciTech Connect

    Studer, Anthony

    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.

  1. Carbonic anhydrases in chick extra-embryonic structures: a role for CA in bicarbonate reabsorption through the chorioallantoic membrane.

    PubMed

    Gabrielli, M Gabriella

    2004-06-01

    The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.

  2. A double origin electrophoretic method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II.

    PubMed

    Murch, R S; Gambel, A M; Kearney, J J

    1986-10-01

    A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods.

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

    NASA Astrophysics Data System (ADS)

    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

  4. Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

    PubMed

    Mogensen, Estelle Geweiss; Janbon, Guilhem; Chaloupka, James; Steegborn, Clemens; Fu, Man Shun; Moyrand, Frédérique; Klengel, Torsten; Pearson, David S; Geeves, Michael A; Buck, Jochen; Levin, Lonny R; Mühlschlegel, Fritz A

    2006-01-01

    Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH. PMID:16400172

  5. Two atypical carbonic anhydrase homologs from the planula larva of the scleractinian coral Fungia scutaria.

    PubMed

    deBoer, Melissa L; Krupp, Dave A; Weis, Virginia M

    2006-08-01

    In cnidarians, the enzyme carbonic anhydrase (CA) is important to inorganic carbon (Ci) flux in processes including calcification and dinoflagellate symbiont photosynthesis. Although CA is known to function in Ci delivery to symbionts in adults with mature symbioses, it is not known when CA becomes active in this capacity during the onset of symbiosis in developing hosts. We identified two CA cDNA sequences from the planula larvae of the Hawaiian scleractinian coral Fungia scutaria. Expression of these larval CAs did not differ between infected and uninfected larvae or vary over the course of infection. Bioinformatic analyses of the two homologs showed that the sequences are unusually short and are missing some residues that support active site structure in other CAs. This is the first description of a short form of CA. Phylogenetic analyses of the larval CAs grouped them with membrane-bound homologs from vertebrates. Studies in other calcifying cnidarians have identified membrane-associated CAs as functioning in calcification, and therefore the two larval CAs could play a role in the onset of calcification during metamorphosis. A longer CA isoform was amplified from adult F. scutaria cDNA but not from larvae, suggesting that the longer form is not expressed in larvae. The longer form grouped with cytosolic CAs including a symbiotic anemone homolog implicated in Ci delivery to dinoflagellate symbionts. The apparent absence of this "symbiosis" CA in larvae suggests that the Ci supply mechanism is not active during the initial onset of the association. PMID:16946238

  6. How to get into bones: proton pump and carbonic anhydrase in Osedax boneworms

    PubMed Central

    Tresguerres, Martin; Katz, Sigrid; Rouse, Greg W.

    2013-01-01

    Osedax are gutless siboglinid worms that thrive on vertebrate bones lying on the ocean floor, mainly those of whales. The posterior body of female Osedax penetrates into the bone forming extensions known as ‘roots’, which host heterotrophic symbiotic bacteria in bacteriocytes beneath the epidermis. The Osedax root epithelium presumably absorbs bone collagen and/or lipids, which are metabolized by the symbiotic bacteria that in turn serve for Osedax's nutrition. Here, we show that Osedax roots express extremely high amounts of vacuolar-H+-ATPase (VHA), which is located in the apical membrane and in cytoplasmic vesicles of root and ovisac epithelial cells. The enzyme carbonic anhydrase (CA), which catalyses the hydration of CO2 into H+ and HCO3−, is also expressed in roots and throughout Osedax body. These results suggest Osedax roots have massive acid-secreting capacity via VHA, fuelled by H+ derived from the CA-catalysed hydration of CO2 produced by aerobic metabolism. We propose the secreted acid dissolves the bone carbonate matrix to then allow the absorption of bone-derived nutrients across the skin. In an exciting example of convergent evolution, this model for acid secretion is remarkably similar to mammalian osteoclast cells. However, while osteoclasts dissolve bone for repairing and remodelling, the Osedax root epithelium secretes acid to dissolve foreign bone to access nutrients. PMID:23760644

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

    PubMed

    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

  8. CO{sub 2} availability, carbonic anhydrase, and the annual dinoflagellate bloom in Lake Kinneret

    SciTech Connect

    Berman-Frank, I; Zohary, T.; Erez, J.

    1994-12-01

    Changes with time in the concentration of inorganic carbon in Lake Kinneret subsurface water were followed throughout two seasonal dinoflagellate blooms. The response of natural populations of the dominant dinoflagellate, Peridinium gatunense, to these changes was recorded by examining fluctuations over time in the activity of the enzyme carbonic anhydrase (CA) and in photosynthetic parameters. Our results show distinct fluctuations of both external and cytoplasmic CA activity in P. gatunense throughout the annual bloom. Higher levels of activity were triggered by the decline of total dissolved inorganic C below 1.8 mM and more specifically by low concentrations of dissolved CO{sub 2} (1-10 {mu}M) during the seasonal bloom decline in May-June. Laboratory studies on cultured P. gatunense confirmed our field observations, suggesting that supplemental mechanisms are activated in P. gatunense that enhance inorganic C uptake when CO{sub 2} is limiting for photosynthesis. Eventually, the cellular adaptations of P. gatunense to the declining CO{sub 2} concentrations could not prevent decline of photosynthetic rates contributing to the subsequent decrease in P. gatunense biomass in May-June. In Lake Kinneret, P. gatunense is succeeded by Peridiniopsis spp., the photosynthetic rates and external CA activities of which were much higher under environmental conditions typical of the end of the bloom. 45 refs., 9 figs., 2 tabs.

  9. Characterization and function of carbonic anhydrases in the zooxanthellae-giant clam symbiosis.

    PubMed

    Baillie, B K; Yellowlees, D

    1998-03-22

    Carbonic anhydrase (CA) has been purified from the host tissue of Tridacna gigas, a clam that lives in symbiosis with the dinoflagellate alga, Symbiodinium. At least two isoforms of CA were identified in both gill and mantle tissue. The larger (70 kDa) isoform is a glycoprotein with both N- and O-glycans attached and has highest homology to CAII. It is associated with the membrane fraction while the smaller (32 kDa) is present in the aqueous phase in both tissues. The 32 kDa CA has high homology with mammalian CAI at the N-terminus. Both isoforms cross-reacted with antibodies to CAII from chicken. Immunohistology demonstrated that the 70 kDa CA is present within the ciliated branchial filaments and cells lining the tertiary water channels in the gills of T. gigas. This is consistent with a role in the transport of inorganic carbon (Ci) to the haemolymph and therefore supply of Ci to the zooxanthellae. CA was also detected in mantle epithelial cells where it may also contribute to Ci supply to the zooxanthellae. The hyaline body and nerve tissue in the mantle express the 70 kDa CA where it may be involved in light sensing and nervous transmission.

  10. Histochemical Localisation of Carbonic Anhydrase in the Inner Ear of developing Cichlid Fish, Oreochromis mossambicus

    NASA Astrophysics Data System (ADS)

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

    Inner ear otolith growth in terms of mineralisation mainly depends on the enzyme carbonic anhydrase CA CA 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 CA was analysed during the early larval development of a teleost the cichlid fish Oreochromis mossambicus CA-reactivity was observed already in stage 7 animals onset of otocyst development staging follows Anken et al Zool Anz 231 1-10 1993 Neuroblasts from which sensory and supporting cells as well as ionocytes are derived proved to be CA positive Already at stage 12 hatch CA-positive regions could be attributed to ionocyte containg 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 CA in the cichlid With the onset of stage 16 finray primordia in dorsal fin yolk-sac being increasingly absorbed CA-reactivity was observed in the vestibular nerve This indicates the onset of myelinisation and thus commencement of operation The localisation of CA in the inner ear of fish especially the differences in comparison to mammals is discussed on the basis of its role in otolith

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

    PubMed

    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

  12. Optimization of a Novel Peptide Ligand Targeting Human Carbonic Anhydrase IX

    PubMed Central

    Rana, Shoaib; Nissen, Felix; Marr, Annabell; Markert, Annette; Altmann, Annette; Mier, Walter; Debus, Juergen; Haberkorn, Uwe; Askoxylakis, Vasileios

    2012-01-01

    Background Carbonic anhydrase IX (CA IX) is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1) was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. Methodology/Principal Findings Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy) was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. Conclusions Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of the ligand in

  13. An experimental study on the effect of carbonic anhydrase on the oxygen isotope exchange kinetics and equilibrium in the carbonic acid system

    NASA Astrophysics Data System (ADS)

    Uchikawa, J.; Zeebe, R. E.

    2011-12-01

    Stable oxygen isotopes of marine biogenic carbonates are often depleted in 18O relative to the values expected for thermodynamic equilibrium with ambient seawater. One possibility is that 18O-depletion in carbonates is kinetically controlled. The kinetic isotope effect associated with the hydration of CO2 results in 18O-depleted HCO3-. If the HCO3- is utilized before re-establishing equilibrium with ambient water under rapid calcification, the 18O-depletion will be recorded in carbonates. But one caveat in this kinetic model is the fact that many marine calcifiers posses carbonic anhydrase, a zinc-bearing enzyme that catalyzes the CO2 hydration reaction. It is expected that this enzyme accelerates 18O-equilibration in the carbonic acid system by facilitating direct oxygen isotope exchange between HCO3- and H2O via CO2 hydration. Clearly this argues against the conceptual framework of the kinetic model. Yet the critical variable here is the effectiveness of the carbonic anhydrase, which is likely to depend on its concentration and the carbonate chemistry of the aqueous medium. It is also hitherto unknown whether the presence of carbonic anhydrase alters the equilibrium oxygen isotope fractionations between dissolved carbonate species and water. We performed a series of quantitative inorganic carbonate precipitation experiments to examine the changes in the oxygen isotope equilibration time as a function of carbonic anhydrase concentrations. We conducted experiments at pH 8.3 and 8.9. These pH values are similar to the average surface ocean pH and the elevated pH levels observed within calcification microenvironments of certain corals and planktonic foraminifera. A summary of our new experimental results will be presented.

  14. Physiological and Molecular Biological Characterization of Intracellular Carbonic Anhydrase from the Marine Diatom Phaeodactylum tricornutum1

    PubMed Central

    Satoh, Dan; Hiraoka, Yasutaka; Colman, Brian; Matsuda, Yusuke

    2001-01-01

    A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO2-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to β-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO2-binding site appeared to be unique among the β-CAs so far reported. PMID:11500545

  15. Quantification of extracellular carbonic anhydrase activity in two marine diatoms and investigation of its role.

    PubMed

    Hopkinson, Brian M; Meile, Christof; Shen, Chen

    2013-06-01

    Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO2) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on (18)O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO2 concentration. In support of a role for eCA in CO2 supply, eCA activity specifically responded to low CO2 rather than to changes in pH or HCO3(-), and the rates of eCA activity are nearly optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Although the CO2 gradients abolished by eCA are small (less than 0.5 μm concentration difference between bulk and cell surface), CO2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO2 gradient. Alternative roles for eCA in CO2 recovery as HCO3(-) and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.

  16. Effect of carbonic anhydrase on silicate weathering and carbonate formation at present day CO₂ concentrations compared to primordial values.

    PubMed

    Xiao, Leilei; Lian, Bin; Hao, Jianchao; Liu, Congqiang; Wang, Shijie

    2015-01-01

    It is widely recognized that carbonic anhydrase (CA) participates in silicate weathering and carbonate formation. Nevertheless, it is still not known if the magnitude of the effect produced by CA on surface rock evolution changes or not. In this work, CA gene expression from Bacillus mucilaginosus and the effects of recombination protein on wollastonite dissolution and carbonate formation under different conditions are explored. Real-time fluorescent quantitative PCR was used to explore the correlation between CA gene expression and sufficiency or deficiency in calcium and CO₂ concentration. The results show that the expression of CA genes is negatively correlated with both CO₂ concentration and ease of obtaining soluble calcium. A pure form of the protein of interest (CA) is obtained by cloning, heterologous expression, and purification. The results from tests of the recombination protein on wollastonite dissolution and carbonate formation at different levels of CO₂ concentration show that the magnitudes of the effects of CA and CO₂ concentration are negatively correlated. These results suggest that the effects of microbial CA in relation to silicate weathering and carbonate formation may have increased importance at the modern atmospheric CO₂ concentration compared to 3 billion years ago.

  17. Effect of carbonic anhydrase on silicate weathering and carbonate formation at present day CO2 concentrations compared to primordial values

    NASA Astrophysics Data System (ADS)

    Xiao, Leilei; Lian, Bin; Hao, Jianchao; Liu, Congqiang; Wang, Shijie

    2015-01-01

    It is widely recognized that carbonic anhydrase (CA) participates in silicate weathering and carbonate formation. Nevertheless, it is still not known if the magnitude of the effect produced by CA on surface rock evolution changes or not. In this work, CA gene expression from Bacillus mucilaginosus and the effects of recombination protein on wollastonite dissolution and carbonate formation under different conditions are explored. Real-time fluorescent quantitative PCR was used to explore the correlation between CA gene expression and sufficiency or deficiency in calcium and CO2 concentration. The results show that the expression of CA genes is negatively correlated with both CO2 concentration and ease of obtaining soluble calcium. A pure form of the protein of interest (CA) is obtained by cloning, heterologous expression, and purification. The results from tests of the recombination protein on wollastonite dissolution and carbonate formation at different levels of CO2 concentration show that the magnitudes of the effects of CA and CO2 concentration are negatively correlated. These results suggest that the effects of microbial CA in relation to silicate weathering and carbonate formation may have increased importance at the modern atmospheric CO2 concentration compared to 3 billion years ago.

  18. Localization and quantification of carbonic anhydrase activity in the symbiotic Scyphozoan Cassiopea xamachana.

    PubMed

    Estes, Anne M; Kempf, Stephen C; Henry, Raymond P

    2003-06-01

    The relationship between density and location of zooxanthellae and levels of carbonic anhydrase (CA) activity was examined in Cassiopea xamachana. In freshly collected symbiotic animals, high densities of zooxanthellae corresponded with high levels of CA activity in host bell and oral arm tissues. Bleaching resulted in a significant loss of zooxanthellae and CA activity. Recolonization resulted in full restoration of zooxanthellar densities but only partial restoration of CA activity. High levels of CA activity were also seen in structures with inherently higher zooxanthellar densities, such as oral arm tissues. Similarly, the oral epidermal layer of bell tissue had significantly higher zooxanthellar densities and levels of CA activity than did aboral bell tissues. Fluorescent labeling, using 5-dimethylaminonapthalene-1-sulfonamide (DNSA) also reflected this tight-knit relationship between the presence and density of zooxanthellae, as DNSA-CA fluorescence intensity was greatest in host oral epithelial cells directly overlying zooxanthellae. However, the presence and density of zooxanthellae did not always correspond with enzyme activity levels. A transect of bell tissue from the margin to the manubrium revealed a gradient of CA activity, with the highest values at the bell margin and the lowest at the manubrium, despite an even distribution of zooxanthellae. Thus, abiotic factors may also influence the distribution of CA and the levels of CA activity. PMID:12807705

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory isoform by sulfonamides.

    PubMed

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

    2009-07-15

    The inhibition of a newly cloned coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of sulfonamides, including some clinically used derivatives (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 topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with low potency inhibitors (K(I)s in the range of 163-770nM), or with medium potency inhibitors (K(I)s in the range of 75.1-105nM), whereas ethoxzolamide, several clinically used sulfonamides and heterocyclic compounds showed stronger potency, with K(I)s in the range of 16-48.2nM. These inhibitors may be useful to better understand the physiological role of the Stylophora pistillata CA (STPCA) in corals and its involvement in biomineralisation in this era of global warming.

  1. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    PubMed

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

    2016-02-15

    The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(6) M(-1) s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K(I) of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. 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, by allowing the identification of inhibitors which may be useful as pharmacologic tools. PMID:26832216

  2. Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: novel sulfamoylcarbamates and sulfamides derived from acetophenones.

    PubMed

    Akıncıoğlu, Akın; Akıncıoğlu, Hülya; Gülçin, İlhami; Durdagi, Serdar; Supuran, Claudiu T; Göksu, Süleyman

    2015-07-01

    In this study, several novel sulfamides were synthesized and evaluated for their acetylcholine esterase (AChE) and human carbonic anhydrase I, and II isoenzymes (hCA I and II) inhibition profiles. Reductive amination of methoxyacetophenones was used for the synthesis of amines. Amines were converted to sulfamoylcarbamates with chlorosulfonyl isocyanate (CSI) in the presence of BnOH. Pd-C catalyzed hydrogenolysis of sulfamoylcarbamates afforded sulfamides. These novel compounds were good inhibitors of the cytosolic hCA I, and hCA II with Ki values in the range of 45.9±8.9-687.5±84.3 pM for hCA I, and 48.80±8.2-672.2±71.9pM for hCA II. The inhibitory effects of the synthesized novel compounds on AChE were also investigated. The Ki values of these compounds were in the range of 4.52±0.61-38.28±6.84pM for AChE. These results show that hCA I, II, and AChE were effectively inhibited by the novel sulfamoylcarbamates 17-21 and sulfamide derivatives 22-26. All investigated compounds were docked within the active sites of the corresponding enzymes revealing the reasons of the effective inhibitory activity. PMID:25921269

  3. Ligand-Induced Protein Mobility in Complexes of Carbonic Anhydrase II and Benzenesulfonamides with Oligoglycine Chains

    PubMed Central

    Krishnamurthy, Vijay M.; Raman, Venkata S.; Mowery, Richard A.; Hentz, Michelle; Baleja, James D.; Shaw, Bryan F.; Kumar, Krishna

    2013-01-01

    This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with 15N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches. PMID:23472094

  4. The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis.

    PubMed

    Mahon, Brian P; Bhatt, Avni; Socorro, Lilien; Driscoll, Jenna M; Okoh, Cynthia; Lomelino, Carrie L; Mboge, Mam Y; Kurian, Justin J; Tu, Chingkuang; Agbandje-McKenna, Mavis; Frost, Susan C; McKenna, Robert

    2016-08-23

    Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes. PMID:27439028

  5. Synthesis and carbonic anhydrase inhibitory effects of novel sulfamides derived from 1-aminoindanes and anilines.

    PubMed

    Akbaba, Yusuf; Bastem, Enes; Topal, Fevzi; Gülçin, Ilhami; Maraş, Ahmet; Göksu, Süleyman

    2014-12-01

    Three 1-aminoindanes, four anilines and BnOH or t-BuOH were reacted with chlorosulfonyl isocyanate to give sulfamoyl carbamates. Pd-C catalysed hydrogenolysis reactions of carbamates or deprotection of the Boc group of the carbamates with CF3 CO2 H afforded seven novel sulfamides. Human carbonic anhydrase (hCA) isoenzymes I and II (hCA I and hCA II) were purified from fresh human blood erythrocytes with one-step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The inhibitory properties of the novel sulfamides on both isoenzymes were determined using the esterase activity with 4-nitrophenyl acetate (NPA) as substrate. The tested novel sulfamides derived from 1-aminoindanes and anilines effectively inhibited hCA I and II competitively in the nanomolar range. Among these compounds, the novel sulfamide derivative 17 showed the most potent inhibitory effect against hCA I (Ki : 153.88 nM), while sulfamide derivative 26 showed the highest inhibitory potential against hCA II (Ki : 117.80 nM). PMID:25223956

  6. Carbonic anhydrase inhibitors: glycosylsulfanilamides act as subnanomolar inhibitors of the human secreted isoform VI.

    PubMed

    Winum, Jean-Yves; Montero, Jean-Louis; Vullo, Daniela; Supuran, Claudiu T

    2009-12-01

    A series of sulfonamides incorporating sugar moieties and the sulfanilamide scaffold have been investigated for their interaction with the secretory isoform of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA VI. This isoform is secreted in saliva, tears, and milk of mammals - where it plays important physiological roles - even if little is understood at this moment regarding its inhibition, due to the lack of potent and/or selective inhibitors. Here we report a series of low nanomolar and subnanomolar CA VI inhibitors, belonging to the glycosylamine-sulfanilamide class. The glucose, ribose, arabinose, xylose, and fucose derivatives showed excellent CA VI inhibitory activity, with K(i)s in the range of 0.56-5.1 nm, whereas the least active derivatives, incorporating gallactose, mannose, and rhamnose scaffolds showed inhibition constants in the range of 10.1-34.1 nm. Many of these sulfonamides were also selective inhibitors for their interaction with CA VI over the physiologically dominant and ubiquitous isoform CA II, with selectivity ratios of 4.11-35.93 for inhibiting the secreted over the cytosolic isozyme. Because of their high water solubility and high affinity for CA VI over CA II, these compounds are useful tools for better understanding the secreted CA isoform CA VI. PMID:19824891

  7. Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors.

    PubMed

    Kaya, Muharrem; Basar, Erhan; Cakir, Emrah; Tunca, Ekrem; Bülbül, Metin

    2012-08-01

    Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, (13)C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of sulfanilamide, acetazolamide (AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been studied in vitro. The IC(50) values of compounds for esterase activity are 0.71-0.11 µM for hCA I and 0.45-0.12 µM for hCA II, respectively. The K(i) values of these inhibitors were determined as 0,38-0,008 µM for hCA I and 0,19-0,001 µM for hCA II, respectively. PMID:21846203

  8. Inhibition of β-carbonic anhydrases with ureido-substituted benzenesulfonamides.

    PubMed

    Pacchiano, Fabio; Carta, Fabrizio; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2011-01-01

    A series of sulfonamides was prepared by reaction of sulfanilamide with aryl/alkyl isocyanates. The ureido-substituted benzenesulfonamides showed a very interesting profile for the inhibition of several carbonic anhydrases (CAs, EC 4.2.1.1) such as the human hCA II and three β-CAs from pathogenic fungal or bacterial species. The Candida albicans enzyme was inhibited with potencies in the range of 3.4-3970 nM, whereas the Mycobacterium tuberculosis enzymes Rv1284 and Rv3273 were inhibited with K(i)s in the range of 4.8-6500 nM and of 6.4-6850 nM, respectively. The structure-activity relationship for this class of inhibitors is rather complex, but the main features associated with effective inhibition of both α- and β-CAs investigated here have been delineated. The nature of the moiety substituting the second ureido nitrogen is the determining factor in controlling the inhibitory power, probably due to the flexibility of the ureido linker and the possibility of this moiety to orientate in different subpockets of the active site cavities of these enzymes. PMID:21145236

  9. Carbonic anhydrase inhibitory properties of novel benzylsulfamides using molecular modeling and experimental studies.

    PubMed

    Göksu, Süleyman; Naderi, Ali; Akbaba, Yusuf; Kalın, Pınar; Akıncıoğlu, Akın; Gülçin, İlhami; Durdagi, Serdar; Salmas, Ramin Ekhteiari

    2014-10-01

    In this study, a series of sulfamoyl carbamates and sulfamide derivatives were synthesized. Six commercially available benzyl amines and BnOH were reacted with chlorosulfonyl isocyanate (CSI) to give sulfamoyl carbamates. Pd-C catalyzed hydrogenolysis reactions of carbamates afforded sulfamides. The inhibition effects of novel benzylsulfamides on the carbonic anhydrase I, and II isoenzymes (CA I, and CA II) purified from fresh human blood red cells were determined by Sepharose-4B-L-Tyrosine-sulfanilamide affinity chromatography. In vitro studies were shown that all of novel synthesized benzylsulfamide analogs inhibited, concentration dependently, both hCA isoenzyme activities. The novel benzylsulfamide compounds investigated here exhibited nanomolar inhibition constants against the two isoenzymes. Ki values were in the range of 28.48±0.01-837.09±0.19nM and 112.01±0.01-268.01±0.22nM for hCAI and hCA II isoenzymes, respectively. Molecular modeling approaches were also applied for studied compounds. PMID:25159522

  10. Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.

    PubMed

    Demirdağ, Ramazan; Çomaklı, Veysel; Şentürk, Murat; Ekinci, Deniz; İrfan Küfrevioğlu, Ö; Supuran, Claudiu T

    2013-03-15

    Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31μM. PMID:22974493

  11. Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III.

    PubMed

    Silverman, D N; Tu, C; Chen, X; Tanhauser, S M; Kresge, A J; Laipis, P J

    1993-10-12

    Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change. PMID:8399223

  12. Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila.

    PubMed Central

    Williams, T. G.; Colman, B.

    1993-01-01

    External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions. PMID:12231991

  13. Genetic polymorphisms in the carbonic anhydrase VI gene and dental caries susceptibility.

    PubMed

    Li, Z-Q; Hu, X-P; Zhou, J-Y; Xie, X-D; Zhang, J-M

    2015-06-01

    We investigated the role of 7 single nucleotide polymorphisms in the carbonic anhydrase (CA) VI gene (rs2274328, rs17032907, rs11576766, rs2274333, rs10864376, rs3765964, and rs6680186) and the possible association between these polymorphisms and dental caries susceptibility in a Northwestern Chinese population. We collected samples from 164 high caries experience and 191 very low caries experience and conducted a case-control study according to the number of decayed, missing, and filled teeth index and genotyped the 7 polymorphisms using a 384-well plate format with the Sequenom MassARRAY platform. Individuals carrying the rs17032907 TT genotype were more likely to have an increased risk of dental caries compared with carriers of the C/C genotype in the co-dominant model, with an odds ratio (95% confidence interval) of 2.144 (1.096-4.195). We also found that the haplotype (ACA) (rs2274328, rs17032907 and rs11576766) was associated with a low number of decayed, missing, and filled teeth index with an odds ratio (95% confidence interval) of 0.635 (0.440-0.918). However, we found no association between dental caries susceptibility and the rs2274328, rs11576766, rs2274333, rs10864376, rs3765964, and rs6680186 polymorphisms and other haplotypes. The rs17032907 genetic variant and the haplotype (ACA) of CA VI may be associated with dental caries susceptibility.

  14. Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

    PubMed Central

    Vilas, Gonzalo; Krishnan, Devishree; Loganathan, Sampath Kumar; Malhotra, Darpan; Liu, Lei; Beggs, Megan Rachele; Gena, Patrizia; Calamita, Giuseppe; Jung, Martin; Zimmermann, Richard; Tamma, Grazia; Casey, Joseph Roman; Alexander, Robert Todd

    2015-01-01

    Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins. PMID:25609088

  15. Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis, molecular docking and anticonvulsant studies.

    PubMed

    Karataş, Mert Olgun; Uslu, Harun; Sarı, Suat; Alagöz, Mehmet Abdullah; Karakurt, Arzu; Alıcı, Bülent; Bilen, Cigdem; Yavuz, Emre; Gencer, Nahit; Arslan, Oktay

    2016-10-01

    Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.

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

    PubMed Central

    Rahim, S. M.; Mazlan, A. G.; Simon, K. D.; Delaunoy, J. P.; Laurent, P.

    2014-01-01

    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 (Oncorhynchus mykiss) to clarify its physiological function. CA was purified from rainbow trout gills O. mykiss and specific antibodies were raised. Immunoblotting between tissue homogenates of pseudobranch and gill CA antibodies showed specific immunostaining with only one band corresponding to CA in the pseudobranch homogenate. Results of immunohistochemical 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 showed that some CA enzyme was cytoplasmic and the remainder was linked to membranous structures. The results also showed that the lacunar tissue layers did not display immunoperoxidase activity. Our results indicated that pseudobranch CA may have a function related to the extracellular medium wherein CA intervenes with the mechanism of stimulation of afferent nerve fibers. PMID:24510712

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

    PubMed

    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.

  18. Spectroscopic and MD simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA induced by urea.

    PubMed

    Idrees, Danish; Prakash, Amresh; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-09-01

    Carbonic anhydrase VA (CAVA) is primarily expressed in the mitochondria and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis and neuronal transmission. To understand the biophysical properties of CAVA, we carried out a reversible urea-induced isothermal denaturation at pH 7.0 and 25°C. Spectroscopic probes, [θ]222 (mean residue ellipticity at 222 nm), F344 (Trp-fluorescence emission intensity at 344 nm) and Δε280 (difference absorption at 280 nm) were used to monitor the effect of urea on the structure and stability of CAVA. The urea-induced reversible denaturation curves were used to estimate [Formula: see text], Gibbs free energy in the absence of urea; Cm, the mid-point of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Coincidence of normalized transition curves of all optical properties suggests that unfolding/refolding of CAVA is a two-state process. We further performed 40 ns molecular dynamics simulation of CAVA to see the dynamics at different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.

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

    PubMed

    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

  20. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    PubMed

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

    2016-02-15

    The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(6) M(-1) s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K(I) of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. 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, by allowing the identification of inhibitors which may be useful as pharmacologic tools.

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

    PubMed

    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.

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

    PubMed

    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

  3. More effective dithiocarbamate derivatives inhibiting carbonic anhydrases, generated by QSAR and computational design.

    PubMed

    Avram, Speranta; Milac, Adina Luminita; Carta, Fabrizio; Supuran, Claudiu T

    2013-04-01

    Dithiocarbamates (DTC) are promising compounds with potential applications in antitumoral and glaucoma therapy. Our aim is to understand molecular features affecting DTC interaction with carbonic anhydrases (CAs), zinc-containing enzymes maintaining acid-base balance in blood and other tissues. To this end, we generate QSAR models based on a compound series containing 25 DTC, inhibitors of four human (h) CAs isoforms: hCA I, II, IX and XII. We establish that critical physicochemical parameters for DTC inhibitory activity are: hydrophobic, electronic, steric, topological and shape. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q(2) (0.55-0.73), fitted correlation r(2) (0.75-0.84) and standard error of prediction (0.47-0.23). Based on the established QSAR equations, we analyse 22 new DTC derivatives and identify DTC dicarboxilic acids derivatives and their esters as potentially improved inhibitors of CA I, II, IX and XII. PMID:23116520

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

    PubMed

    Rahim, S M; Mazlan, A G; Simon, K D; Delaunoy, J P; Laurent, P

    2014-02-01

    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 (Oncorhynchus mykiss) to clarify its physiological function. CA was purified from rainbow trout gills O. mykiss and specific antibodies were raised. Immunoblotting between tissue homogenates of pseudobranch and gill CA antibodies showed specific immunostaining with only one band corresponding to CA in the pseudobranch homogenate. Results of immunohistochemical 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 showed that some CA enzyme was cytoplasmic and the remainder was linked to membranous structures. The results also showed that the lacunar tissue layers did not display immunoperoxidase activity. Our results indicated that pseudobranch CA may have a function related to the extracellular medium wherein CA intervenes with the mechanism of stimulation of afferent nerve fibers. PMID:24510712

  5. Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms.

    PubMed

    Syrjänen, Leo; Luukkaala, Tiina; Leppilampi, Mari; Kallioinen, Matti; Pastorekova, Silvia; Pastorek, Jaromir; Waheed, Abdul; Sly, William S; Parkkila, Seppo; Karttunen, Tuomo

    2014-09-01

    Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours.

  6. The Evolutionary History of Daphniid α-Carbonic Anhydrase within Animalia

    PubMed Central

    Culver, Billy W.; Morton, Philip K.

    2015-01-01

    Understanding the mechanisms that drive acid-base regulation in organisms is important, especially for organisms in aquatic habitats that experience rapidly fluctuating pH conditions. Previous studies have shown that carbonic anhydrases (CAs), a family of zinc metalloenzymes, are responsible for acid-base regulation in many organisms. Through the use of phylogenetic tools, this present study attempts to elucidate the evolutionary history of the α-CA superfamily, with particular interest in the emerging model aquatic organism Daphnia pulex. We provide one of the most extensive phylogenies of the evolution of α-CAs, with the inclusion of 261 amino acid sequences across taxa ranging from Cnidarians to Homo sapiens. While the phylogeny supports most of our previous understanding on the relationship of how α-CAs have evolved, we find that, contrary to expectations, amino acid conservation with bacterial α-CAs supports the supposition that extracellular α-CAs are the ancestral state of animal α-CAs. Furthermore, we show that two cytosolic and one GPI-anchored α-CA in Daphnia genus have homologs in sister taxa that are possible candidate genes to study for acid-base regulation. In addition, we provide further support for previous findings of a high rate of gene duplication within Daphnia genus, as compared with other organisms. PMID:25893130

  7. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  8. Immobilization of carbonic anhydrase on carboxyl-functionalized ferroferric oxide for CO2 capture.

    PubMed

    Lv, Bihong; Yang, Zhaoren; Pan, Fujun; Zhou, Zuoming; Jing, Guohua

    2015-08-01

    New materials of Fe3O4 magnetic microspheres were functionalized with carboxyl and prepared for carbonic anhydrase (CA) immobilization to capture CO2. The optimum conditions for immobilization, such as carrier dose, enzyme dose, pH, shaking speed, temperature and contact time, were determined. The pH and thermal stability of the free and the immobilized CA were compared. The results presented that the immobilized CA had a better enzyme activity, a higher pH and thermal stability than that of the free CA. Meanwhile, CO2 capture was respectively enhanced by the free and the immobilized CA in tris(hydroxymethyl) aminomethane (Tris) buffer solution. Moreover, the immobilized CA maintained 58.5% of its initial catalytic ability even after ten recovery cycles due to the protest of the magnetic microspheres. All the results confirmed the potential use of the carboxyl-functionalized Fe3O4 magnetic microspheres immobilized CA to remove CO2 from air or flue gas.

  9. GdnHCl-induced unfolding intermediate in the mitochondrial carbonic anhydrase VA.

    PubMed

    Idrees, Danish; Prakash, Amresh; Haque, Md Anzarul; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2016-10-01

    Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25°C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25M to 3.40M of the denaturant. However, CAVA gets completely denatured at 4.0M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40ns were carried out and a well-defined correlation was established for both in vitro and in silico studies. PMID:27365118

  10. Using electrospray ionization FTICR mass spectrometry to study competitive binding of inhibitors to carbonic anhydrase

    SciTech Connect

    Cheng, X.; Chen, R.; Bruce, J.E.; Schwartz, B.L.; Anderson, G.A.; Hofstadler, S.A.; Gale, D.C.; Smith, R.D.; Gao, J.; Sigal, G.B.; Mammen, M.; Whitesides, G.M.

    1995-08-30

    We report a method based on mass spectrometry for the characterization of noncovalent complexes of proteins with mixtures of ligands; this method is relevant to the study of drug leads and may be useful in screening libraries for tight-binding compounds. This study describes the competitive binding of inhibitors derived from para-substituted benzenesulfonamides to bovine carbonic anhydrase II (BCAII, EC 4.2.1.1) using this technique. Relative binding constants and structural information for a mixture of inhibitors can be obtained in a single experiment using ESI-FTICR-MS. The work demonstrates that ESI-MS has significant potential for measuring relative binding affinities and characterizing the structures of ligands associated noncovalently to proteins. We have detected noncovalent complexes in the gas phase for ligands having values of K{sub b} as low as 1.7 x 10{sup 6} M{sup -1} in solution. The technique also allowed identification of tightbinding ligands from small libraries. The structures of inhibitors having similar masses can be identified by the high-resolution and multistep dissociation mass spectrometry of which FTICR is uniquely capable. This range of capabilities for ESI-FTICR-MS should be widely useful in medicinal chemistry. 22 refs., 2 figs.

  11. Postnatal disappearance of type A intercalated cells in carbonic anhydrase II-deficient mice.

    PubMed

    Brion, L P; Suarez, C; Saenger, P

    2001-06-01

    Despite chronic acidosis, collecting ducts in adult carbonic anhydrase II-deficient (CAD mice) are depleted of intercalated cells, including those of type A, which are acid-secreting cells. We hypothesized that this depletion could occur during postnatal development. Principal cells were identified by immunofluorescence using an antibody to rat aquaporin-2 (AQP-2), and type A intercalated cells using an antibody specific for anion exchanger (AE1). In CAD mice the proportion of AQP2-positive cells, normal at 11 days, increased progressively in the cortical (CCD) and outer medullary collecting duct (OMCD), to reach almost 100% in the OMCD in adults. The percentage of AE1-positive cells in the OMCD of CAD mice decreased by half by 6 weeks of age and further by adulthood. In controls, however, the proportion of AQP2-positive cells and that of AE1-positive cells in the OMCD remained stable after 10 days of age. AE1-positive cells accounted for the majority of intercalated cells in the OMCD. The mechanisms leading to selective postnatal cell depletion in the collecting duct in CAD mice remain to be determined.

  12. Influence of pesticide exposure on carbonic anhydrase II from sheep stomach.

    PubMed

    Kılınç, Namık; İşgör, Mehmet Mustafa; Şengül, Bülent; Beydemir, Şükrü

    2015-09-01

    Carbonic anhydrase (CA) is a widely distributed enzyme and has a crucial role in the cells, tissues and organs of living organisms. It is found that CA-II is one of the most abundant CA isoenzymes in the gastrointestinal system. It plays an important role in the gastric acid secretion in stomach. In this study, we purified CA-II isoenzyme from sheep stomach with a 615.2 purification fold, 78% purification yield and 5562.02 specific activity. Moreover, the in vitro effects of some commonly used pesticides including chlorpyrifos, cypermethrin, dichlorvos, glyphosate isopropylamine and lambda cyhalomethrin on the enzyme activity were investigated. Of these compounds, glyphosate isopropylamine and dichlorvos showed an inhibition on CA-II esterase activity. They have IC50 values of 0.155 µM and 2.690 µM and Ki values of 0.329 µM and 3.654 µM, respectively. Both glyphosate isopropylamine and dichlorvos inhibited CA-II isoenzyme in a noncompetitive manner.

  13. Building reactive copper center(s) in human carbonic anhydrase II

    PubMed Central

    Song, He; Weitz, Andrew C.; Hendrich, Michael P.; Lewis, Edwin A.; Emerson, Joseph P.

    2014-01-01

    Re-engineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here we report thermodynamic data characterizing the formation of two type-2 (T2) copper sites in carbonic anhydrase and experimental evidence showing one of these new copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and in enzymatic systems. Human CA II is known to bind two Cu2+ ions; herein, these binding events are explored using modern isothermal titration calorimetry (ITC) techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (Ka ∼ 5 × 1012 and 1 × 1010) and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site, and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol. PMID:23744511

  14. Immobilized Carbonic Anhydrase on Hollow Fiber Membranes Accelerates CO2 Removal from Blood

    PubMed Central

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

    2012-01-01

    Current artificial lungs and respiratory assist devices designed for carbon dioxide removal (CO2R) are limited in their efficiency due to the relatively small partial pressure difference across gas exchange membranes. To offset this underlying diffusional challenge, bioactive hollow fiber membranes (HFMs) increase the carbon dioxide diffusional gradient through the immobilized enzyme carbonic anhydrase (CA), which converts bicarbonate to CO2 directly at the HFM surface. In this study, we tested the impact of CA-immobilization on HFM CO2 removal efficiency and thromboresistance in blood. Fiber surface modification with radio frequency glow discharge (RFGD) introduced hydroxyl groups, which were activated by 1M CNBr while 1.5M TEA was added drop wise over the activation time course, then incubation with a CA solution covalently linked the enzyme to the surface. The bioactive HFMs were then potted in a model gas exchange device (0.0084 m2) and tested in a recirculation loop with a CO2 inlet of 50mmHg under steady blood flow. Using an esterase activity assay, CNBr chemistry with TEA resulted in 0.99U of enzyme activity, a 3.3 fold increase in immobilized CA activity compared to our previous method. These bioactive HFMs demonstrated 108 ml/min/m2 CO2 removal rate, marking a 36% increase compared to unmodified HFMs (p < 0.001). Thromboresistance of CA-modified HFMs was assessed in terms of adherent platelets on surfaces by using lactate dehydrogenase (LDH) assay as well as scanning electron microscopy (SEM) analysis. Results indicated HFMs with CA modification had 95% less platelet deposition compared to unmodified HFM (p < 0.01). Overall these findings revealed increased CO2 removal can be realized through bioactive HFMs, enabling a next generation of more efficient CO2 removal intravascular and paracorporeal respiratory assist devices. PMID:22962517

  15. Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

    PubMed

    De Cian, Marie-Cécile; Bailly, Xavier; Morales, Julia; Strub, Jean-Marc; Van Dorsselaer, Alain; Lallier, François H

    2003-05-15

    The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed. PMID:12696045

  16. Asymmetric subcellular mRNA distribution correlates with carbonic anhydrase activity in Acetabularia acetabulum.

    PubMed

    Serikawa, K A; Porterfield, D M; Mandoli, D F

    2001-02-01

    The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. CO2 bioconversion using carbonic anhydrase (CA): effects of PEG rigidity on the structure of bio-mineralized crystal composites.

    PubMed

    Hwang, Ee Taek; Gang, Haemin; Gu, Man Bock

    2013-10-20

    The combined effect of both carbonic anhydrase (CA) and the rigidity of polyethylene glycol (PEG) were found to assist the bio-mineralized crystallization behavior of CO2 differentially. In this study, different forms of magnetically responsive calcium carbonate (CaCO3) crystal composites were successfully formed from gaseous CO2 by using the different forms of polyethylene glycols (PEGs) in a constant CO2 pressure controlled chamber. Polygonal particles were produced with more rigid polymer chains (branched PEG), whereas less rigid polymer chains (PEG) induced the formation of ellipsoidal particles. However, no morphological changes occurred without the presence of CA.

  19. Evidence that an internal carbonic anhydrase is present in 5% CO/sub 2/-grown and air-grown Chlamydomonas. [Chlamydomonas reinhardtii

    SciTech Connect

    Moroney, J.V.; Togasaki, R.K.; Husic, H.D.; Tolbert, N.E.

    1987-07-01

    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 CO/sub 2/. Both air-grown cells, that have a CO/sub 2/ concentrating system, and 5% CO/sub 2/-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% CO/sub 2/-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 CO/sub 2/ fixation by high CO/sub 2/-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO/sub 2/-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.

  20. Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air▿ †

    PubMed Central

    Burghout, Peter; Cron, Lorelei E.; Gradstedt, Henrik; Quintero, Beatriz; Simonetti, Elles; Bijlsma, Jetta J. E.; Bootsma, Hester J.; Hermans, Peter W. M.

    2010-01-01

    The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO2) it encounters during transmission, colonization, and infection. Since CO2 is important for various cellular processes, factors that allow optimal CO2 sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO2-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO2 to bicarbonate (HCO3−), an essential step to prevent the cellular release of CO2. The addition of unsaturated fatty acids (UFAs) reversed the CO2-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO2 fixation is at least associated with HCO3−-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO2 fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO2-independent and naturally circulating CO2-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO2 fixation pathways for pneumococcal growth and survival. PMID:20525828

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

    PubMed

    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

  2. Protonography, a new technique for the analysis of carbonic anhydrase activity.

    PubMed

    De Luca, Viviana; Del Prete, Sonia; Supuran, Claudiu T; Capasso, Clemente

    2015-04-01

    All proteolytic enzymes, which are able to renature and reacquire the proteolytic activity on a copolymerized substrate, can be analyzed by zymography upon removal of sodium dodecyl sulfate (SDS). Protonography, the new technique described in this study, unlike zymography, allows the detection of a different protein, not a protease, i.e. of the carbonic anhydrase (CA, EC 4.2.1.1) activity on a SDS polyacrylamide gel electrophoresis gel. CAs are zinc-containing enzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate and protons. Hydrogen ions produced during the catalyzed reaction are responsible for the change of color that appears on the gel around the CA band. For this reason, we named the new technique "protonography". The following four salient features characterize this new technique: (a) on the basis of molecular weight markers, recombinant or native CAs with different molecular weights can be detected and quantified rapidly on a single gel; (b) the hydratase activity can be reversibly inhibited by SDS during electrophoresis and recovered by incubating the gel in aqueous Triton X-100; (c) it is possible to separate active oligomeric forms of CAs on the gel enabling their activities to be determined independently of one another. This feature is not possible when using solution assays; and (d) it can be a useful tool to establish if a putative or a newly identified CA in a genome is expressed and enzymatically active. This article outlines the general principles employed in protonography, providing an easy procedure to implement it in laboratories working with CAs. It also presents an overview of its development and current research applications through specific examples. PMID:25007132

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Suitability of the alkalistable carbonic anhydrase from a polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic carbon sequestration.

    PubMed

    Bose, Himadri; Satyanarayana, T

    2016-10-01

    Carbonic anhydrase (CA) was produced from the polyextremophilic (halotolerant, moderately thermophilic and alkaliphilic) bacterium Aeribacillus pallidus TSHB1 isolated from water and sediment samples of Choti Anhoni hot spring of Pipariya, Madhya Pradesh (India), is being reported to be suitable for carbon sequestration. Growth and CA production were inhibited at higher CO2 concentration (5-10 %). Under optimized culture variables (tryptone 0.8 %, yeast extract 0.08 %, glucose 1 %, micronutrient solution 1 %, inoculums size 1.10 %, agitation 200 at pH 8, and temperature 55 °C), 3.7-fold higher CA production was attained than that under unoptimized conditions. The zymogram analysis of the partially purified CA revealed an activity band corresponding to 32 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 with T 1/2 of 40, 15, and 8 min at 60, 70, and 80 °C, respectively. The CA of A. pallidus displayed a marked enhancement in the rate of CaCO3 precipitation from aqueous CO2. The CA-aided formation of CaCO3 was 42.5 mg mg(-1) protein. Scanning electron microscopy revealed the formation of rhomboid calcite crystals. This is the first report on the production and applicability of CA from the polyextremophilic A. pallidus in carbon sequestration. PMID:27215773

  5. Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO2-Requiring Phenotype 1

    PubMed Central

    Price, G. D.; Badger, M. R.

    1989-01-01

    Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (Ci) and low Ci grown cells leads the creation of a high Ci requiring phenotype causing: (a) a dramatic increase in the K0.5 (Ci) for photosynthesis, (b) a loss of the ability to accumulate internal Ci, and (c) a decrease in the lag between the initial Ci accumulation following illumination and the efflux of CO2 from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO2 concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and Ci accumulation is developed in which the carboxysome plays a central role as both the site of CO2 generation from HCO3− and a resistance barrier to CO2 efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells. Images Figure 7 PMID:16667062

  6. Enzyme-accelerated and structure-guided crystallization of calcium carbonate: role of the carbonic anhydrase in the homologous system.

    PubMed

    Müller, Werner E G; Schlossmacher, Ute; Schröder, Heinz C; Lieberwirth, Ingo; Glasser, Gunnar; Korzhev, Michael; Neufurth, Meik; Wang, Xiaohong

    2014-01-01

    The calcareous spicules from sponges, e.g. from Sycon raphanus, are composed of almost pure calcium carbonate. In order to elucidate the formation of those structural skeletal elements, the function of the enzyme carbonic anhydrase (CA), isolated from this species, during the in vitro calcium carbonate-based spicule formation, was investigated. It is shown that the recombinant sponge CA substantially accelerates calcium carbonate formation in the in vitro diffusion assay. A stoichiometric calculation revealed that the turnover rate of the sponge CA during the calcification process amounts to 25 CO2s(-1) × molecule CA(-1). During this enzymatically driven process, initially pat-like particles are formed that are subsequently transformed to rhomboid/rhombohedroid crystals with a dimension of ~50 μm. The CA-catalyzed particles are smaller than those which are formed in the absence of the enzyme. The Martens hardness of the particles formed is ~4 GPa, a value which had been determined for other biogenic calcites. This conclusion is corroborated by energy-dispersive X-ray spectroscopy, which revealed that the particles synthesized are composed predominantly of the elements calcium, oxygen and carbon. Surprising was the finding, obtained by light and scanning electron microscopy, that the newly formed calcitic crystals associate with the calcareous spicules from S. raphanus in a highly ordered manner; the calcitic crystals almost perfectly arrange in an array orientation along the two opposing planes of the spicules, leaving the other two plane arrays uncovered. It is concluded that the CA is a key enzyme controlling the calcium carbonate biomineralization process, which directs the newly formed particles to existing calcareous spicular structures. It is expected that with the given tools new bioinspired materials can be fabricated.

  7. In Vitro Evaluation of ESE-15-ol, an Estradiol Analogue with Nanomolar Antimitotic and Carbonic Anhydrase Inhibitory Activity

    PubMed Central

    Stander, Barend Andre; Joubert, Fourie; Tu, Chingkuang; Sippel, Katherine H.; McKenna, Robert; Joubert, Annie Margaretha

    2012-01-01

    Antimitotic compounds are still one of the most widely used chemotherapeutic anticancer drugs in the clinic today. Given their effectiveness against cancer it is beneficial to continue enhancing these drugs. One way is to improve the bioavailability and efficacy by synthesizing derivatives that reversibly bind to carbonic anhydrase II (CAII) in red blood cells followed by a slow release into the blood circulation system. In the present study we describe the in vitro biological activity of a reduced derivative of 2-ethyl-3-O-sulphamoyl-estradiol (2EE), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol). ESE-15-ol is capable of inhibiting carbonic anhydrase activity in the nanomolar range and is selective towards a mimic of carbonic anhydrase IX when compared to the CAII isoform. Docking studies using Autodock Vina suggest that the dehydration of the D-ring plays a role towards the selectivity of ESE-15-ol to CAIX and that the binding mode of ESE-15-ol is substantially different when compared to 2EE. ESE-15-ol is able to reduce cell growth to 50% after 48 h at 50–75 nM in MCF-7, MDA-MB-231, and MCF-12A cells. The compound is the least potent against the non-tumorigenic MCF-12A cells. In vitro mechanistic studies demonstrate that the newly synthesized compound induces mitochondrial membrane depolarization, abrogates the phosphorylation status of Bcl-2 and affects gene expression of genes associated with cell death and mitosis. PMID:23300615

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

    SciTech Connect

    Hu, P.Y.; Ernst, A.R.; Sly, W.S. ); Venta, P.J. ); Skaggs, L.A.; Tashian, R.E. )

    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.

  9. Permeability changes of cationic liposomes loaded with carbonic anhydrase induced by millimeter waves radiation.

    PubMed

    Di Donato, Loreto; Cataldo, Maria; Stano, Pasquale; Massa, Rita; Ramundo-Orlando, Alfonsina

    2012-11-01

    The interaction of millimeter wave radiation, in the 30-300 GHz range, with biological systems is a topic of great interest as many of the vibrational dynamics that occur in biochemical reactions of large macromolecules in living organisms fall in the 1-100 GHz range. Membranes and cellular organelles may have different ways of interacting with this radiation as well. In this article, we investigate the influence of 53.37 GHz of radiation on lipid membrane permeability by using cationic liposomes that contain dipalmitoylphosphatidylcholine (DPPC), cholesterol and stearylamine. Carbonic anhydrase (CA) is loaded inside the liposome and the substrate p-nitrophenyl acetate (p-NPA) is added in the bulk aqueous phase. Upon permeation across the lipid bilayer, the trapped CA catalyzes the conversion of the p-NPA molecules into products. Because the self-diffusion rate of p-NPA across intact liposomes is very low, the CA reaction rate expressed as ΔA/min is used to track membrane permeability changes. A highly significant (P < 0.0001) enhancement of the CA reaction rate, typically from ΔA/min = 0.0043 ± 0.0017 (n = 26) to ΔA/min = 0.0100 ± 0.0020 (n = 32) resulted at a low-level density power of 0.1 mW/cm(2). The enhancement of the CA reaction rate was observed at a lesser extent on liposomes with a larger diameter and, in turn with leaflets less bent. The different packing of the phospholipid bilayer-due to the higher curvature-could be a critical factor in eliciting membrane permeability changes indicating a possible role for water molecules bound to functional groups in the glycerol region. Since numerical dosimetry indicates that the temperature rise during the exposure was negligible, the observed effects cannot be attributed to heating of the samples.

  10. Inhibition of mammalian carbonic anhydrase isoforms I-XIV with a series of phenolic acid esters.

    PubMed

    Maresca, Alfonso; Akyuz, Gulay; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T

    2015-11-15

    A series of phenolic acid esters incorporating caffeic, ferulic, and p-coumaric acid, and benzyl, m/p-hydroxyphenethyl- as well as p-hydroxy-phenethoxy-phenethyl moieties were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Many of the mammalian isozymes of human (h) or murine (m) origin, hCA I-hCA XII, mCA XIII and hCA XIV, were inhibited in the submicromolar range by these derivatives (with KIs of 0.31-1.03 μM against hCA VA, VB, VI, VII, IX and XIV). The off-target, highly abundant isoforms hCA I and II, as well as hCA III, IV and XII were poorly inhibited by many of these esters, although the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, possess a CA inhibition mechanism distinct of the sulfonamides/sulfamates, clinically used drugs for the treatment of a multitude of pathologies, but with severe side effects due to hCA I/II inhibition. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule, binding more externally within the active site cavity, and making contacts with amino acid residues at the entrance of the active site. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds shows isoform-selective inhibitory profiles, which may be exploited for obtaining pharmacological agents with less side effects compared to other classes of inhibitors. PMID:26498394

  11. Carbonic anhydrase enzymes II, VII, IX and XII in colorectal carcinomas

    PubMed Central

    Viikilä, Pia; Kivelä, Antti J; Mustonen, Harri; Koskensalo, Selja; Waheed, Abdul; Sly, William S; Pastorek, Jaromir; Pastorekova, Silvia; Parkkila, Seppo; Haglund, Caj

    2016-01-01

    AIM To investigate expression of four alpha-carbonic anhydrases (CAs) in colorectal carcinomas (CRC) and compare the results with patients’ survival. METHODS Colorectal carcinoma samples from 539 CRC patients and control tissues were arranged as tissue microarrays and analyzed with antibodies against CA II, CA VII, CA IX, and CA XII. Intensity and extent of staining were both scored from 0 to 3 in each sample. These enzyme expression levels were then correlated to patients’ survival and clinicopathological parameters, which were tumor differentiation grade and stage, site of tumor, patients’ age, and gender. Kaplan-Meier analysis and Cox regression hazard ratio model were used to analyze survival data. RESULTS CA II and CA XII staining intensities correlated with patients’ survival in that higher expression indicated poorer prognosis. In Cox regression analysis one unit increase in the CA II intensity increased the hazard ratio to 1.19 fold (CI: 1.04-1.37, P = 0.009). A significant correlation was also found when comparing CA XII staining intensity with survival of CRC patients (HR = 1.18, 95%CI: 1.01-1.38, P = 0.036). The extent of CA XII immunostaining did not correlate to the patients’ survival (P = 0.242, Kaplan-Meier analysis). A significant interaction between age group and extent of the CA II staining was found. Increased extent of CA II had a significant hazard ratio among patients 65 years and older (1.42, 95%CI: 1.16-1.73, P = 0.0006). No correlations were found between CA VII (intensity P = 0.566, extent P = 0.495, Kaplan-Meier analysis), or CA IX (intensity P = 0.879, extent P = 0.315, Kaplan-Meier analysis) immunostaining results and survival, or the other parameters. CONCLUSION The present findings indicate that CA II and CA XII could be useful in predicting survival in CRC. PMID:27688658

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

    PubMed Central

    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 measured VA/Q relationships before and after CA inhibition by the multiple inert gas elimination technique. Four other groups of dogs were studied to control for possible confounding effects of time under anesthesia and nonselective CA inhibition by acetazolamide: (a) saline placebo as a control for duration of anesthesia, (b) 4% CO2 inhalation to mimic systemic CO2 retention, (c) 1 mg/kg benzolamide (a selective renal CA inhibitor) or 0.5 meq/kg HCl to mimic systemic metabolic acidosis, and (d) 500 mg/kg 4,4'-dinitrostilbene-2,2'-disulfonate (an inhibitor of red cell band 3 protein) to mimic the respiratory acidosis arising from an intracapillary block to rapid mobilization of plasma HCO3- in CO2 exchange. Acetazolamide increased VA/Q mismatch and reduced arterial PO2 measured at equilibrium but these did not occur in the control group. There was no deterioration in VA/Q matching when systemic respiratory acidosis produced either by CO2 inhalation or 4,4'-dinitrostilbene-2,2'-disulfonate or metabolic acidosis (benzolamide or HCl) were imposed to mimic the effects of acetazolamide apart from its inhibition of lung CA. These results support the concept that lung CA subserves VA/Q matching in the normal lung. Images PMID:8349809

  13. Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM

    SciTech Connect

    Peña, Kerry L.; Castel, Stephane E.; de Araujo, Charlotte; Espie, George S.; Kimber, Matthew S.

    2010-04-26

    Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO{sub 2} by carbonic anhydrase (CA) prior to fixation. Paradoxically, many {beta}-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal {beta}-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to {gamma}-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA - the first catalytically active bacterial {gamma}-CA reported. The 2.0 {angstrom} structure of CcmM209 reveals a trimeric, left-handed {beta}-helix structure that closely resembles Cam, except that residues 198-207 form a third {alpha}-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 {angstrom} structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the {beta}-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.

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

    PubMed

    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

  15. Effects of Carbonyl Sulfide and Carbonic Anhydrase on Stomatal Conductance1[OA

    PubMed Central

    Stimler, Keren; Berry, Joseph A.; Yakir, Dan

    2012-01-01

    The potential use of carbonyl sulfide (COS) as tracer of CO2 flux into the land biosphere stimulated research on COS interactions with leaves during gas exchange. We carried out leaf 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, using mid-infrared laser spectroscopy. A narrow range in the normalized ratio of the net uptake rates of COS (As) and CO2 (Ac), leaf relative uptake (As/Ac × [CO2]/[COS]), was observed, with a mean value of 1.61 ± 0.26, which is advantageous to the use of COS in photosynthesis research. Notably, increasing COS concentrations between 250 and 2,800 pmol mol−1 (enveloping atmospheric levels) enhanced stomatal conductance (gs) to a variable extent in most plants examined (up to a normalized enhancement factor [ fe = (gs-max − gs-min)/gs-min] of 1). This enhancement was completely abolished in carbonic anhydrase (CA)-deficient antisense lines of both C3 and C4 plants. We suggest that the stomatal response is mediated by CA and may involve hydrogen sulfide formed in the reaction of COS and water with CA. In all species examined, the uptake rates of COS and CO2 were highly correlated, but there was no relationship between the sensitivity of stomata to COS and the rate of COS uptake (or, by inference, hydrogen sulfide production). The basis for the observed stomatal sensitivity and its variations is still to be determined. PMID:22106096

  16. Effect of carbonic anhydrase inhibition on superficial and deep nephron bicarbonate reabsorption in the rat.

    PubMed Central

    DuBose, T D; Lucci, M S

    1983-01-01

    The nephron segment responsible for the acetazolamide-insensitive fraction of renal bicarbonate reabsorption has not been clearly delineated. This study compares superficial and deep nephron bicarbonate reabsorption before and after acetazolamide at two dose levels (20 and 50 mg/kg per h) in mutant Munich-Wistar rats employing both cortical and papillary micropuncture and microcalorimetry. Systemic acid-base balance and right whole kidney glomerular filtration rate were similar in all groups examined. The effects of the two doses of acetazolamide were indistinguishable and resulted in a significant increase in whole kidney bicarbonate excretion that compared favorably with the fraction delivered out of the left papillary tip. Acetazolamide inhibited superficial proximal bicarbonate reabsorption by 80.0%, whereas reabsorption up to the deep loop of Henle was decreased by only 52% (P less than 0.001). Bicarbonate reabsorption that was insensitive to acetazolamide occurred in the superficial and deep loop of Henle and between the distal tubule and base collecting duct. Because water reabsorption in these segments could serve to generate transepithelial bicarbonate concentration gradients favorable for reabsorption, we attempted to minimize water abstraction by combined administration of mannitol and acetazolamide. During this condition a significant increase in bicarbonate delivery up to the deep loop of Henle was noted (52 vs. 65%), whereas superficial nephron reabsorption was not altered. Furthermore, an outwardly directed bicarbonate concentration gradient from the deep loop of Henle to vasa recta was demonstrated during acetazolamide (delta tCO2 = 20.9 +/- 3.3 mM), but was abolished during combined mannitol and acetazolamide administration (delta tCO2 = 3.5 +/- 0.9 mM). It is concluded that carbonic anhydrase inhibition results in a disparate effect on nephron bicarbonate reabsorption when juxtamedullary and superficial nephron segments are compared. Our findings

  17. In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II.

    PubMed

    Bayram, Esra; Senturk, Murat; Kufrevioglu, O Irfan; Supuran, Claudiu T

    2008-10-15

    The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and II, with a series of salicylic acid derivatives was investigated by using the esterase method with 4-nitrophenyl acetate as substrate. IC(50) values for sulfasalazine, diflunisal, 5-chlorosalicylic acid, dinitrosalicylic acid, 4-aminosalicylic acid, 4-sulfosalicylic acid, 5-sulfosalicylic acid, salicylic acid, acetylsalicylic acid (aspirin) and 3-metylsalicylic acid were of 3.04 microM, 3.38 microM, 4.07 microM, 7.64 microM, 0.13 mM, 0.29 mM, 0.42 mM, 0.56 mM, 2.71 mM and 3.07 mM for hCA I and of 4.49 microM, 2.70 microM, 0.72 microM, 2.80 microM, 0.75 mM, 0.72 mM, 0.29 mM, 0.68 mM, 1.16 mM and 4.70 mM for hCA II, respectively. Lineweaver-Burk plots were also used for the determination of the inhibition mechanism of these substituted phenols, most of which were noncompetitive inhibitors with this substrate. Some salicylic acid derivatives investigated here showed effective hCA I and II inhibitory activity, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.

  18. Inhibition of carbonic anhydrases I and II by N-unsubstituted carbamate esters.

    PubMed

    Parr, J S; Khalifah, R G

    1992-12-15

    We previously showed that the zinc metalloenzyme carbonic anhydrases (CA I and II isozymes) bind "neutral" amides and related compounds as anions through coordination of their deprotonated amide nitrogen to the active site zinc (Rogers, J. I., Mukherjee, J., and Khalifah, R. G. (1987) Biochemistry 26, 5672-5679). Urethan, the ethyl carbamate ester, was among such compounds. The present study was designed to test whether other N-unsubstituted carbamate esters of pharmacological interest (as sedatives, hypnotics, anxiolytics, and skeletal muscle relaxants) were capable of binding to CA in the same manner. We studied the interaction of human CA I and II with urethan, phenyl carbamate, ethinamate, meprobamate, and methocarbamol. Phenyl carbamate studies were greatly complicated by its uncatalyzed hydrolysis via an elimination mechanism to form cyanate, a powerful CA inhibitor. In general, the compounds display: 1) slow on-off inhibition binding kinetics in the seconds range, 2) maximal inhibitor affinity at alkaline pH, and 3) characteristic three-band visible spectra of their complexes with cobalt-substituted CA I. These properties are shared with the previously studied amide inhibitors and are taken as evidence that the deprotonated carbamate nitrogen coordinates to the active site metal ion. CA I appeared to bind carbamate esters more tightly than CA II, an unusual 1000-fold selectivity being seen in the case of methocarbamol. The inhibition by these drugs is not sufficiently strong to implicate CA I and II in their mechanism of action. However, it does suggest the possible existence of previously unsuspected similarities between binding to CA and to their physiological receptors or targets, particularly the involvement of zinc. PMID:1460006

  19. Functional characterization of neuroendocrine regulation of branchial carbonic anhydrase induction in the euryhaline crab Callinectes sapidus.

    PubMed

    Mitchell, Reed T; Henry, Raymond P

    2014-12-01

    Carbonic anhydrase (CA) plays an essential role as a provider of counterions for Na(+)/H(+) and Cl(-)/HCO3 (-) exchange in branchial ionic uptake processes in euryhaline crustaceans. CA activity and gene expression are low in crabs acclimated to full-strength seawater, with transfer to low salinity resulting in large-scale inductions of mRNA and subsequent enzyme activity in the posterior ion-regulating gills (e.g., G7). In the green crab Carcinus maenas, CA has been shown to be under inhibitory neuroendocrine control by a putative hormone in the x-organ-sinus gland complex (XOSG), located in the eyestalk. This study characterizes the neuroendocrine regulation of CA induction in the blue crab Callinectes sapidus, a commonly used experimental organism for crustacean osmoregulation. In crabs acclimated to full-strength seawater, eyestalk ligation (ESL) triggered a 1.8- and 100-fold increase in CA activity and mRNA, respectively. Re-injection with eyestalk homogenates abolished increases in CA activity and fractionally reduced CA gene expression. ESL also enhanced CA induction by 33% after 96 h in crabs transferred to 15 ppt salinity. Injection of eyestalk homogenates into intact crabs transferred from 35 to 15 ppt diminished by 43% the CA induction stimulated by low salinity. These results point to the presence of a repressor hormone in the eyestalk. Separate injections of medullary tissue (MT) and sinus gland (SG), two components of the eyestalk, reduced salinity-stimulated CA activity by 22% and 49%, suggesting that the putative repressor is localized to the SG. Crabs injected with SG extract harvested from crabs acclimated to 5 ppt showed no decrease in CA activity, demonstrating that the hormone is down-regulated at low salinity. Our results show the presence in the XOSG of an inhibitory compound that regulates salinity-stimulated CA induction. PMID:25572216

  20. Carbonic anhydrase induction in euryhaline crustaceans is rate-limited at the post-transcriptional level.

    PubMed

    Mitchell, Reed T; Henry, Raymond P

    2014-03-01

    The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25 ppt, respectively) for 7 days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6h; whereas it took 48 h for the initial increases in CA activity (120% and 31%), and 4 to 7 days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15 ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5 ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake. PMID:24333600

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

    PubMed

    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

  2. Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm*

    PubMed Central

    Wandernoth, Petra M; Mannowetz, Nadja; Szczyrba, Jaroslaw; Grannemann, Laura; Wolf, Anne; Becker, Holger M.; Sly, William S.; Wennemuth, Gunther

    2015-01-01

    HCO3− is a key factor in the regulation of sperm motility. High concentrations of HCO3− in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3−, represent potential candidates in the regulation of the HCO3− homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3− homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3−-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3− or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3−. These results suggest that CAII and CAIV are required for optimal fertilization. PMID:26487715

  3. Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

    PubMed

    Esbaugh, A J; Secor, S M; Grosell, M

    2015-09-01

    Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy.

  4. Isoaspartate, Carbamoyl phosphate synthase-1, and Carbonic anhydrase-III as biomarkers of liver injury

    PubMed Central

    Carter, Wayne G.; Vigneswara, Vasanthy; Newlaczyl, Anna; Wayne, Declan; Ahmed, Bilal; Saddington, Stephen; Brewer, Charlotte; Raut, Nikhilesh; Gerdes, Henry K.; Erdozain, Amaia M.; Tooth, David; Bolt, Edward L.; Osna, Natalie A.; Tuma, Dean J.; Kharbanda, Kusum K.

    2015-01-01

    We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ~75-80 kDa, ~95-100 kDa, and ~155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ~160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (~2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as

  5. Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients

    PubMed Central

    Kalavska, Katarina; Chovanec, Michal; Zatovicova, Miriam; Takacova, Martina; Gronesova, Paulina; Svetlovska, Daniela; Baratova, Magdalena; Miskovska, Vera; Obertova, Jana; Palacka, Patrik; Rajec, Jan; Sycova-Mila, Zuzana; Cierna, Zuzana; Kajo, Karol; Spanik, Stanislav; Babal, Pavel; Mardiak, Jozef; Pastorekova, Silvia; Mego, Michal

    2016-01-01

    Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics. PMID:27698832

  6. Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm.

    PubMed

    Wandernoth, Petra M; Mannowetz, Nadja; Szczyrba, Jaroslaw; Grannemann, Laura; Wolf, Anne; Becker, Holger M; Sly, William S; Wennemuth, Gunther

    2015-12-01

    HCO3 (-) is a key factor in the regulation of sperm motility. High concentrations of HCO3 (-) in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3 (-), represent potential candidates in the regulation of the HCO3 (-) homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3 (-) homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3 (-)-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3 (-) or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3 (-). These results suggest that CAII and CAIV are required for optimal fertilization.

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

    PubMed Central

    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, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site specific mutations intended to disrupt 4MI binding have demonstrated these sites to be non-productive. In the present work MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway PMID:21452838

  8. Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients

    PubMed Central

    Kalavska, Katarina; Chovanec, Michal; Zatovicova, Miriam; Takacova, Martina; Gronesova, Paulina; Svetlovska, Daniela; Baratova, Magdalena; Miskovska, Vera; Obertova, Jana; Palacka, Patrik; Rajec, Jan; Sycova-Mila, Zuzana; Cierna, Zuzana; Kajo, Karol; Spanik, Stanislav; Babal, Pavel; Mardiak, Jozef; Pastorekova, Silvia; Mego, Michal

    2016-01-01

    Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.

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

    PubMed

    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 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 future single or

  10. A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals.

    PubMed

    Kleinke, Tanja; Wagner, Siegfried; John, Harald; Hewett-Emmett, David; Parkkila, Seppo; Forssmann, Wolf-Georg; Gros, Gerolf

    2005-02-01

    We have collected gastrointestinal, mainly colonic, mucus from humans, guinea pigs, rats, and normal and carbonic anhydrase II (CAII)-deficient mice. In the mucus of all species, substantial CA activity was present. Using antibodies against human CA isoforms we found that the human mucus CA differs from cytosolic CAI and CAII, membrane-bound CAIV, and the secreted CAVI of saliva. The high sensitivity of mucus CA to acetazolamide rules out its identity with cytosolic CAIII. Partial sequences obtained from the purified human mucus CA show similarity, but not identity, with human CAI, and clear differences from the other known CAs. Additional evidence concerning the CA isoform present in mucus was obtained for the mucus CA of other species and was derived from: (1) the mucus of CAII-deficient mice, whose high CA activity confirms that it is not CAII that is responsible; (2) the inhibitory effect of iodide, which shows that mucus CA from mice, guinea pig and humans does not have the high anion sensitivity of CAI; (3) the inactivating effect of 0.2% SDS on guinea pig, mouse and human mucus CA, ruling out the SDS-resistant CAIV; and (4) the partitioning of guinea-pig mucus CA into the water phase in Triton X114 phase separation experiments, which also argues against its identity with membrane-bound CAs, such as CAIV. A comparison of colonic mucus CA activity in normal and germ-free rats indicates that the mucus CA is not of bacterial origin but is produced by the gastrointestinal tissues. We conclude (from its immunoreactivity, from iodide inhibition and from partial amino acid sequences) that mucus CA of human origin probably represents an isozyme, which is specific for mucus and is not identical with the known CA isozymes. The results obtained for mucus CA of other species collectively point in the same direction. PMID:15671337

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

    PubMed

    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

  12. Carbonic anhydrase enzymes II, VII, IX and XII in colorectal carcinomas

    PubMed Central

    Viikilä, Pia; Kivelä, Antti J; Mustonen, Harri; Koskensalo, Selja; Waheed, Abdul; Sly, William S; Pastorek, Jaromir; Pastorekova, Silvia; Parkkila, Seppo; Haglund, Caj

    2016-01-01

    AIM To investigate expression of four alpha-carbonic anhydrases (CAs) in colorectal carcinomas (CRC) and compare the results with patients’ survival. METHODS Colorectal carcinoma samples from 539 CRC patients and control tissues were arranged as tissue microarrays and analyzed with antibodies against CA II, CA VII, CA IX, and CA XII. Intensity and extent of staining were both scored from 0 to 3 in each sample. These enzyme expression levels were then correlated to patients’ survival and clinicopathological parameters, which were tumor differentiation grade and stage, site of tumor, patients’ age, and gender. Kaplan-Meier analysis and Cox regression hazard ratio model were used to analyze survival data. RESULTS CA II and CA XII staining intensities correlated with patients’ survival in that higher expression indicated poorer prognosis. In Cox regression analysis one unit increase in the CA II intensity increased the hazard ratio to 1.19 fold (CI: 1.04-1.37, P = 0.009). A significant correlation was also found when comparing CA XII staining intensity with survival of CRC patients (HR = 1.18, 95%CI: 1.01-1.38, P = 0.036). The extent of CA XII immunostaining did not correlate to the patients’ survival (P = 0.242, Kaplan-Meier analysis). A significant interaction between age group and extent of the CA II staining was found. Increased extent of CA II had a significant hazard ratio among patients 65 years and older (1.42, 95%CI: 1.16-1.73, P = 0.0006). No correlations were found between CA VII (intensity P = 0.566, extent P = 0.495, Kaplan-Meier analysis), or CA IX (intensity P = 0.879, extent P = 0.315, Kaplan-Meier analysis) immunostaining results and survival, or the other parameters. CONCLUSION The present findings indicate that CA II and CA XII could be useful in predicting survival in CRC.

  13. Carbonic anhydrase (Nce103p): an essential biosynthetic enzyme for growth of Saccharomyces cerevisiae at atmospheric carbon dioxide pressure.

    PubMed

    Aguilera, Jaime; Van Dijken, Johannes P; De Winde, Johannes H; Pronk, Jack T

    2005-10-15

    The NCE103 gene of the yeast Saccharomyces cerevisiae encodes a CA (carbonic anhydrase) that catalyses the interconversion of CO2 and bicarbonate. It has previously been reported that nce103 null mutants require elevated CO2 concentrations for growth in batch cultures. To discriminate between 'sparking' effects of CO2 and a CO2 requirement for steady-state fermentative growth, we switched glucose-limited anaerobic chemostat cultures of an nce103 null mutant from sparging with pure CO2 to sparging with nitrogen gas. This switch resulted in wash-out of the biomass, demonstrating that elevated CO2 concentrations are required even under conditions where CO2 is produced at high rates by fermentative sugar metabolism. Nutritional analysis of the nce103 null mutant demonstrated that growth on glucose under a non-CO2-enriched nitrogen atmosphere was possible when the culture medium was provided with L-aspartate, fatty acids, uracil and L-argininine. Thus the main physiological role of CA during growth of S. cerevisiae on glucose-ammonium salts media is the provision of inorganic carbon for the bicarbonate-dependent carboxylation reactions catalysed by pyruvate carboxylase, acetyl-CoA carboxylase and CPSase (carbamoyl-phosphate synthetase). To our knowledge, the present study represents the first full determination of the nutritional requirements of a CA-negative organism to date. PMID:15948716

  14. Discovery of low nanomolar and subnanomolar inhibitors of the mycobacterial beta-carbonic anhydrases Rv1284 and Rv3273.

    PubMed

    Güzel, Ozlen; Maresca, Alfonso; Scozzafava, Andrea; Salman, Aydin; Balaban, Alexandru T; Supuran, Claudiu T

    2009-07-01

    A series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides has been derivatized by reaction with 2,4,6-trimethylpyrylium perchlorate, leading to pyridinium derivatives. The new sulfonamides were evaluated as inhibitors of two beta-carbonic anhydrases (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, Rv1284 and Rv3273. The whole series showed excellent nanomolar inhibitory activity, with several subnanomolar inhibitors being detected. Rv1284 and Rv3273 have potential for developing antimycobacterial agents with an alternate mechanism of action. PMID:19438226

  15. A review of the pharmacology of carbonic anhydrase inhibitors for the treatment of glaucoma in dogs and cats.

    PubMed

    Maślanka, Tomasz

    2015-03-01

    Glaucoma is a heterogeneous group of disorders usually associated with elevated intraocular pressure (IOP), leading to optic nerve damage, retinal ganglion cell death and irreversible vision loss. Therefore, medications that lower IOP are the mainstay of glaucoma therapy. Carbonic anhydrase inhibitors (CAIs) are some of the principal drugs used in the management of canine and feline glaucoma. This paper summarises current knowledge of the mechanism of action of these agents and their effect on IOP in dogs and cats. It also discusses potential harmful side effects of CAIs and presents current opinions about their role and place in the medical management of glaucoma in small animals.

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

    PubMed

    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

  17. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

    PubMed

    Andersson, Marlene; Chen, Gefei; Otikovs, Martins; Landreh, Michael; Nordling, Kerstin; Kronqvist, Nina; Westermark, Per; Jörnvall, Hans; Knight, Stefan; Ridderstråle, Yvonne; Holm, Lena; Meng, Qing; Jaudzems, Kristaps; Chesler, Mitchell; Johansson, Jan; Rising, Anna

    2014-08-01

    Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation. PMID:25093327

  18. Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains

    PubMed Central

    Otikovs, Martins; Landreh, Michael; Nordling, Kerstin; Kronqvist, Nina; Westermark, Per; Jörnvall, Hans; Knight, Stefan; Ridderstråle, Yvonne; Holm, Lena; Meng, Qing; Jaudzems, Kristaps; Chesler, Mitchell; Johansson, Jan; Rising, Anna

    2014-01-01

    Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation. PMID:25093327

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

    NASA Astrophysics Data System (ADS)

    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

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

    NASA Astrophysics Data System (ADS)

    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

  1. Surface Engineering of Polypropylene Membranes with Carbonic Anhydrase-Loaded Mesoporous Silica Nanoparticles for Improved Carbon Dioxide Hydration.

    PubMed

    Yong, Joel K J; Cui, Jiwei; Cho, Kwun Lun; Stevens, Geoff W; Caruso, Frank; Kentish, Sandra E

    2015-06-01

    Carbonic anhydrase (CA) is a native enzyme that facilitates the hydration of carbon dioxide into bicarbonate ions. This study reports the fabrication of thin films of active CA enzyme onto a porous membrane substrate using layer-by-layer (LbL) assembly. Deposition of multilayer films consisting of polyelectrolytes and CA was monitored by quartz crystal microgravimetry, while the enzymatic activity was assayed according to the rates of p-nitrophenylacetate (p-NPA) hydrolysis and CO2 hydration. The fabrication of the films onto a nonporous glass substrate showed CO2 hydration rates of 0.52 ± 0.09 μmol cm(-2) min(-1) per layer of bovine CA and 2.6 ± 0.7 μmol cm(-2) min(-1) per layer of a thermostable microbial CA. The fabrication of a multilayer film containing the microbial CA on a porous polypropylene membrane increased the hydration rate to 5.3 ± 0.8 μmol cm(-2) min(-1) per layer of microbial CA. The addition of mesoporous silica nanoparticles as a film layer prior to enzyme adsorption was found to increase the activity on the polypropylene membranes even further to a rate of 19 ± 4 μmol cm(-2) min(-1) per layer of microbial CA. The LbL treatment of these membranes increased the mass transfer resistance of the membrane but decreased the likelihood of membrane pore wetting. These results have potential application in the absorption of carbon dioxide from combustion flue gases into aqueous solvents using gas-liquid membrane contactors. PMID:25984966

  2. Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

    NASA Technical Reports Server (NTRS)

    Hall, G. E.; Kenny, A. D.

    1985-01-01

    The calvaria of 5-to-6-day-old mice treated with 1 x 10 to the -8th M of 1,25(OH)2D3 in vitro for 48 hours are examined in order to study the function of carbonic anhydrase in bone resorption. Calcium concentrations in the culture were measured to assess bone resorption. It is observed that 1,25(OH)2D3 effectively stimulates bone resorption in vitro and the resorption is dose-dependent. The effects of azetazolamide on 1,25(OH)2D3-induced bone resorption are investigated. The data reveal that 1,25(OH)2D3-induced calcium release is associated with an increase in the carbonic anhydrase activity of bone, and bone alkaline phosphatase activity is decreased and acid phosphatase activity is increased in response to 1,25(OH)2D3. A two-fold mechanism for 1,25(OH)2D3-induced bone resorption is proposed; the first mechanism is an indirect activation of osteoclasts and the second involves an interaction between hormone and osteoclast precursors.

  3. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    SciTech Connect

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

    2013-05-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

  4. The carbonic anhydrase of the Chinese crab Eriocheir sinensis: Effects of adaption from tap to salt water

    NASA Astrophysics Data System (ADS)

    Olsowski, A.; Putzenlechner, M.; Böttcher, K.; Graszynski, K.

    1995-03-01

    In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crab Eriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na+/K+-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na+/K+-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.

  5. Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker.

    PubMed

    Azevedo-Linhares, Maristela; Freire, Carolina A

    2015-12-01

    In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present. PMID:26410193

  6. Renal carbonic anhydrase in the quail Coturnix coturnix japonica: I. Activity and distribution in male and female metanephros.

    PubMed

    Gabrielli, M G; Palatroni, P; Vincenzetti, S

    1990-11-01

    Carbonic anhydrase activity was studied in the quail metanephros by means of histochemical, histophotometrical and biochemical methods. Male and female samples were examined separately in order to show sex-related differences in enzyme activity and localization. The staining patterns revealed differential distribution of reaction product in the different tubular segments. The initial portion of proximal tubules showed positivity on the brush border in female kidneys only. Extra situ investigations provided further evidence of sexual dimorphism resulting in higher values of enzyme activity for female than for male kidneys. In both sexes, marked staining was detected at the distal tubule level where histophotometric analysis confirmed the highest amount of reaction product. Moreover, the intracellular staining distribution at this site proved to be similar to that observed for mammalian proximal convoluted tubules. In the collecting ducts, a mosaic-like pattern was found with respect to both carbonic anhydrase staining and metachromatic properties. The functional significance of the presence of enzyme in the different renal tubules is discussed by comparison with the mammalian kidney. A model is proposed whereby the distal tubules represent the main sites of urinary acidification and bicarbonate reabsorption.

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

    SciTech Connect

    Boone, Christopher D.; Tu, Chingkuang; McKenna, Robert

    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.

  8. Design, synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII.

    PubMed

    D'Ascenzio, Melissa; Carradori, Simone; De Monte, Celeste; Secci, Daniela; Ceruso, Mariangela; Supuran, Claudiu T

    2014-03-15

    A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with KIs ranging between 11 and 390 nM, whereas they were inactive against both CA I (KIs >50 μM) and II (K(I)s ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.

  9. Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker.

    PubMed

    Azevedo-Linhares, Maristela; Freire, Carolina A

    2015-12-01

    In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present.

  10. Epithelial carbonic anhydrases facilitate PCO2 and pH regulation in rat duodenal mucosa

    PubMed Central

    Mizumori, Misa; Meyerowitz, Justin; Takeuchi, Tetsu; Lim, Shu; Lee, Paul; Supuran, Claudiu T; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2006-01-01

    The duodenum is the site of mixing of massive amounts of gastric H+ with secreted HCO3−, generating CO2 and H2O accompanied by the neutralization of H+. We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H+ is neutralized, CO2 is absorbed, and HCO3− is secreted. Rat duodena were perfused with solutions of different pH and PCO2 with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and PCO2 electrodes simultaneously measured perfusate and effluent pH and PCO2. High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, PCO2 ≈ 0) accompanied by portal venous (PV) acidification and PCO2 increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3− gain, whereas all CA inhibitors inhibited PV acidification and PCO2 increase. The changes in luminal and PV pH and [CO2] were also inhibited by the Na+–H+ exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO2 output and increased H+ loss with PV acidification and PCO2 increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO2 buffer, loss of CO2 from the lumen was CA dependent as was transepithelial transport of perfused 13CO2. H+ and CO2 loss from the perfusate were accompanied by increases of PV H+ and tracer CO2, but unchanged PV total CO2, consistent with CA-dependent transmucosal H+ and CO2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO3− secretion. Luminal H+ traverses the apical membrane as CO2, is converted back to cytosolic H+, which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO3− into the lumen and CO2 diffusion into duodenal mucosa, serving as important acid–base regulators. PMID:16556652

  11. Evaluating the role of carbonic anhydrases in the transport of HCO3−-related species

    PubMed Central

    Boron, Walter F.

    2015-01-01

    The soluble enzyme carbonic anhydrase II (CAII) plays an important role in CO2 influx and efflux by red blood cells (RBCs), a process initiated by changes in the extracellular [CO2] (CO2-initiated CO2 transport). Evidence suggests that CAII may be part of a macromolecular complex at the inner surface of the RBC membrane. Some have suggested CAII specifically binds to a motif on the cytoplasmic C terminus (Ct) of the Cl–HCO3 exchanger AE1 and some other members of the SLC4 family of HCO3− transporters, a transport metabolon. Moreover, others have suggested that this bound CAII enhances the transport of HCO3−-related species—HCO3−, CO3=, or CO3= ion pairs—when the process is initiated by altering the activity of the transporter (HCO3−-initiated HCO3− transport). In this review, I assess the theoretical roles of CAs in the transport of CO2 and HCO3−-related species, concluding that although the effect of bound CAII on CO2-initiated CO2 transport is expected to be substantial, the effect of bound CAs on HCO3−-initiated HCO3− transport is expected to be modest at best. I also assess the experimental evidence for CAII binding to AE1 and other transporters, and the effects of this binding on HCO3−-initiated HCO3− transport. The early conclusion that CAII binds to the Ct of AE1 appears to be the result of unpredictable effects of GST in the GST fusion proteins used in the studies. The early conclusion that bound CAII speeds HCO3−-initiated HCO3− transport appears to be the result of CAII accelerating the pH changes used as a read-out of transport. Thus, it appears that CAII does not bind directly to AE1 or other SLC4 proteins, and that bound CAII does not substantially accelerate HCO3−-initiated HCO3− transport. PMID:19879980

  12. Prognostic Significance of Carbonic Anhydrase IX Expression in Cancer Patients: A Meta-Analysis

    PubMed Central

    van Kuijk, Simon J. A.; Yaromina, Ala; Houben, Ruud; Niemans, Raymon; Lambin, Philippe; Dubois, Ludwig J.

    2016-01-01

    Hypoxia is a characteristic of many solid tumors and an adverse prognostic factor for treatment outcome. Hypoxia increases the expression of carbonic anhydrase IX (CAIX), an enzyme that is predominantly found on tumor cells and is involved in maintaining the cellular pH balance. Many clinical studies investigated the prognostic value of CAIX expression, but most have been inconclusive, partly due to small numbers of patients included. The present meta-analysis was therefore performed utilizing the results of all clinical studies to determine the prognostic value of CAIX expression in solid tumors. Renal cell carcinoma was excluded from this meta-analysis due to an alternative mechanism of upregulation. 958 papers were identified from a literature search performed in PubMed and Embase. These papers were independently evaluated by two reviewers and 147 studies were included in the analysis. The meta-analysis revealed strong significant associations between CAIX expression and all endpoints: overall survival [hazard ratio (HR) = 1.76, 95% confidence interval (95%CI) 1.58–1.98], disease-free survival (HR = 1.87, 95%CI 1.62–2.16), locoregional control (HR = 1.54, 95%CI 1.22–1.93), disease-specific survival (HR = 1.78, 95%CI 1.41–2.25), metastasis-free survival (HR = 1.82, 95%CI 1.33–2.50), and progression-free survival (HR = 1.58, 95%CI 1.27–1.96). Subgroup analyses revealed similar associations in the majority of tumor sites and types. In conclusion, these results show that patients having tumors with high CAIX expression have higher risk of locoregional failure, disease progression, and higher risk to develop metastases, independent of tumor type or site. The results of this meta-analysis further support the development of a clinical test to determine patient prognosis based on CAIX expression and may have important implications for the development of new treatment strategies. PMID:27066453

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

    PubMed Central

    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

  14. A highly selective space-folded photo-induced electron transfer fluorescent probe for carbonic anhydrase isozymes IX and its applications for biological imaging.

    PubMed

    Zhang, Shenyi; Yang, Chunmei; Lu, Weiqiang; Huang, Jin; Zhu, Weiping; Li, Honglin; Xu, Yufang; Qian, Xuhong

    2011-08-01

    The first highly selective and sensitive fluorescent probe Z1 for detection of carbonic anhydrase IX (CA IX) over isoforms CA I and CA II was developed. As demonstrated, Z1 worked effectively in both enzymatic systems and living hypoxia cells.

  15. A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.

    PubMed Central

    Kisker, C; Schindelin, H; Alber, B E; Ferry, J G; Rees, D C

    1996-01-01

    A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold. This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life. Images PMID:8665839

  16. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

    ERIC Educational Resources Information Center

    Ship, Noam J.; Zamble, Deborah B.

    2005-01-01

    The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

  17. Conversion of carbon dioxide to oxaloacetate using integrated carbonic anhydrase and phosphoenolpyruvate carboxylase.

    PubMed

    Chang, Kwang Suk; Jeon, Hancheol; Gu, Man Bock; Pack, Seung Pil; Jin, EonSeon

    2013-12-01

    The development and implementation of strategies for CO2 mitigation are necessary to counteract the greenhouse gas effect of carbon dioxide emissions. To demonstrate the possibility of simultaneously capturing CO2 and utilizing four-carbon compounds, an integrated system using CA and PEPCase was developed, which mimics an in vivo carbon dioxide concentration mechanism. We first cloned the PEPCase 1 gene of the marine diatom Phaeodactylum tricornutum and produced a recombinant PtPEPCase 1. The affinity column purified PtPEPCase 1 exhibited specific enzymatic activity (5.89 U/mg). When the simultaneous and coordinated reactions of CA from Dunaliella sp. and the PtPEPCase 1 occurred, more OAA was produced than when only PEPCase was present. Therefore, this integrated CA-PEPCase system can be used not only to capture CO2 but also for a new technology to produce value-added four-carbon platform chemicals.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

    PubMed Central

    Elleuche, Skander; Pöggeler, Stefanie

    2009-01-01

    Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3−) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions. PMID:19365544

  20. Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase.

    PubMed

    Guo, C; Gynn, M; Chang, T M S

    2015-06-01

    We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4, or 6 times that of RBC.

  1. Role of cations, anions and carbonic anhydrase in fluid transport across rabbit corneal endothelium

    PubMed Central

    Fischbarg, J.; Lim, J. J.

    1974-01-01

    1. A small electrical potential difference (541 ± 48 μV, aqueous side negative) across rabbit corneal endothelium has been recently found. Its dependence on ambient [Na+], [K+], [H+] and metabolic and specific inhibitors was examined. 2. Changes in concentration of the ions above either were known or were presently shown to affect the rate of fluid transport across this preparation (normal value: 5·2 ± 0·4 μl./hr.cm2). Ionic concentration changes were also found here to influence potential difference in the same way as fluid transport. In the cases tested, the effects on both fluid transport and potential difference were reversible. 3. Fluid transport and potential difference were both decreased or abolished in absence of Na+, K+ and HCO3-, and when [H+] was decreased. Fluid transport and potential difference were saturable functions of [HCO3-] and half-saturation occurred in both cases at about 13 mM-HCO3-. The potential difference was also a saturable function of [Na+] (half-saturation around 15 mM). There was a pH optimum for potential difference in the range 7·4-7·6. Lower pH values decreases the potential difference and the fluid transport, and a small (-100 μV) reversed potential was observed in the range of 5·3-5·5. 4. Total replacement of Cl- by HCO3- or SO42- produced no impairment on either fluid transport or potential difference. 5. Carbonic anhydrase inhibitors (ethoxyzolamide 10-5 or 10-4 M and benzolamide 10-3 M) produced a 40-60% decrease in the rate of fluid pumping. In contrast, ethoxyzolamide 10-4 M or acetazolamide 10-3 M did not produce any change in the potential difference. NaCN and Na iodoacetate (both 2 mM) eliminated the potential difference in 1-1·5 hr while in controls it lasted for 5-6 hr. 6. Ouabain (10-5 M) abolished the potential difference in less than 10 sec when added to the aqueous side, which suggests the existence of an electrogenic pump. This extremely fast time transient can be accounted for by the accessibility

  2. Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila.

    PubMed Central

    Williams, T. G.; Colman, B.

    1995-01-01

    An equation has been developed incorporating whole-cell rate constants for CO2 and HCO3- that describes accurately photosynthesis (Phs) in suspensions of unicellular algae at low dissolved inorganic carbon. At pH 8.0 the concentration of CO2 available to the algal cells depends on the rate of supply from, and the loss to, HCO3- and the rate of use by the cells. At elevated cell densities (>30 mg chlorophyll [Chl] L-1), at which CO2 use by the cells is high, the slope of a graph of absolute Phs versus Chl concentration approaches the rate of Phs on a milligram of Chl basis because of HCO3- use alone. The slope of a graph of Phs versus HCO3- will be the rate constant for HCO3-, and for Chlorella saccharophila it was 0.16 L mg-1 Chl h-1. The difference between the constants for dissolved inorganic carbon (measured in cells with external carbonic anhydrase) and HCO3-1 is the constant for CO2, which was 26 L mg-1 Chl h-1. This difference causes the half-saturation constant for Phs to increase 5- to 6-fold at high cell densities. The increase in CO2 use as a result of external carbonic anhydrase is described mathematically as a function of cell density. PMID:12228358

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

    PubMed Central

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

    2015-01-01

    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

  4. Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I, II and IX with some hydroxylic compounds.

    PubMed

    Durdagi, Serdar; Korkmaz, Neslihan; Işık, Semra; Vullo, Daniela; Astley, Demet; Ekinci, Deniz; Salmas, Ramin E; Senturk, Murat; Supuran, Claudiu T

    2016-12-01

    A series of hydroxylic compounds (1-10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068-4003, 0.012-9.9 and 0.025-115 μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the KI values were calculated.

  5. Inhibition of human carbonic anhydrase isoforms I-XIV with sulfonamides incorporating fluorine and 1,3,5-triazine moieties.

    PubMed

    Ceruso, Mariangela; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2013-11-15

    Reaction of cyanuryl fluoride with sulfanilamide or 4-aminoethylbenzenesulfonamide afforded triazinyl-substituted benzenesulfonamides incorporating fluorine, which were further derivatized by reaction with amines, amino alcohols, amino acids or amino acid esters. Inhibition studies of all the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, hCA I-XIV with these compounds revealed that they show moderate-weak inhibition of hCA III, IV, VA and XIII, rather moderate inhibition against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II, VII and XII. The inhibition profile of these fluorine containing triazinyl sulfonamides is thus very different from the corresponding analogs incorporating chlorine, which were previously investigated as inhibitors of some of these enzymes. PMID:24103430

  6. Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I, II, IX and XII.

    PubMed

    Durgun, Mustafa; Turkmen, Hasan; Ceruso, Mariangela; Supuran, Claudiu T

    2016-03-01

    Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. 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 (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space. PMID:26803577

  7. Carbonic anhydrase inhibition for the management of cerebral ischemia: in vivo evaluation of sulfonamide and coumarin inhibitors.

    PubMed

    Di Cesare Mannelli, Lorenzo; Micheli, Laura; Carta, Fabrizio; Cozzi, Andrea; Ghelardini, Carla; Supuran, Claudiu T

    2016-12-01

    Ischemia of brain areas is a global health problem, causing death or long-term disability. Current pharmacological options have limited impact on ischemic damages. Recently, a relationship between hypoxia and carbonic anhydrase (CA) over-expression has been highlighted suggesting CA inhibition as a possible target. This study aimed to evaluate the pharmacological profile of sulfonamide and coumarin CA inhibitors in rats underwent permanent middle cerebral artery occlusion (pMCAO). The neurological score of pMCAO rats was dramatically reduced 24 h after occlusion. Repeated subcutaneous injections of the CA inhibitors 4 and 7 (1 mg kg(-1)) were able to increase the neurological score by 40%. Compound 7 showed the tendency to reduce the volume of hemisphere infarction. The standard CA inhibitor acetazolamide was ineffective. The properties of novel CA inhibitors to improve neurological functionalities after cerebral ischemic insult are shown. The CA involvement in cerebral hypoxic phenomena deserves deeper investigations.

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

    NASA Technical Reports Server (NTRS)

    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.

  9. The Potential of Liposomes with Carbonic Anhydrase IX to Deliver Anticancer Ingredients to Cancer Cells in Vivo

    PubMed Central

    Ng, Huei Leng Helena; Lu, Aiping; Lin, Ge; Qin, Ling; Yang, Zhijun

    2014-01-01

    Drug delivery nanocarriers, especially targeted drug delivery by liposomes are emerging as a class of therapeutics for cancer. Early research results suggest that liposomal therapeutics enhanced efficacy, while simultaneously reducing side effects, owing to properties such as more targeted localization in tumors and active cellular uptake. Here, we highlight the features of immunoliposomes that distinguish them from previous anticancer therapies, and describe how these features provide the potential for therapeutic effects that are not achievable with other modalities. While a large number of studies has been published, the emphasis here is placed on the carbonic anhydrase IX (CA-IX) and the conjugated liposomes that are likely to open a new chapter on drug delivery system by using immunoliposomes to deliver anticancer ingredients to cancer cells in vivo. PMID:25547490

  10. Effects of Silica Nanoparticle Supported Ionic Liquid as Additive on Thermal Reversibility of Human Carbonic Anhydrase II

    PubMed Central

    Fallahbagheri, Azadeh; Saboury, Ali Akbar; Ma'mani, Leila; Taghizadeh, Mohammad; Khodarahmi, Reza; Ranjbar, Samira; Bohlooli, Mousa; Shafiee, Abbas; Foroumadi, Alireza; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2013-01-01

    Silica nanoparticle supported imidazolium ionic liquid [SNImIL] was synthesized and utilized as a biocompatible additive for studying the thermal reversibility of human carbonic anhydrase II (HCA II). For this purpose, we prepared additive by modification of nanoparticles through the grafting of ionic liquids on the surface of nanoparticles (SNImIL). The SNImIL were fully characterized by Fourier Transform Infrared spectroscopy, scanning electron microscopy and thermo gravimetric analysis. The characterization of HCA II was investigated by various techniques including UV–Vis and ANS fluorescence spectrophotometry, differential scanning calorimetry, and docking study. SNImIL induced disaggregation, enhanced protein stability and increased thermal reversibility of HCA II by up to 42% at pH 7.75. PMID:22829053

  11. Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors.

    PubMed

    Winum, Jean-Yves; Supuran, Claudiu T

    2015-04-01

    In addition to the sulfonamides and their isosteres, recently novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) which act by binding to the metal ion from the active site were discovered. Based on the X-ray crystal structure of the CA II-trithiocarbonate adduct, dithiocarbamates, xanthates and thioxanthates were shown to potently inhibit α- and β-CAs. The hydroxamates constitute another class of recently studied CAIs both against mammalian and protozoan enzymes. Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates, whereas the xanthates and salicylaldoximes were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates showed potent antiglaucoma activity in animal models of the disease whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA.

  12. Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I, II and IX with some hydroxylic compounds.

    PubMed

    Durdagi, Serdar; Korkmaz, Neslihan; Işık, Semra; Vullo, Daniela; Astley, Demet; Ekinci, Deniz; Salmas, Ramin E; Senturk, Murat; Supuran, Claudiu T

    2016-12-01

    A series of hydroxylic compounds (1-10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068-4003, 0.012-9.9 and 0.025-115 μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the KI values were calculated. PMID:26634620

  13. Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

    PubMed

    Gao, J; Cheng, X; Chen, R; Sigal, G B; Bruce, J E; Schwartz, B L; Hofstadler, S A; Anderson, G A; Smith, R D; Whitesides, G M

    1996-05-10

    This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development. PMID:8642553

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

    PubMed

    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. Kinetic and in silico studies of hydroxy-based inhibitors of carbonic anhydrase isoforms I and II.

    PubMed

    Ekhteiari Salmas, Ramin; Mestanoglu, Mert; Durdagi, Serdar; Sentürk, Murat; Kaya, A Afşin; Kaya, Elif Çelenk

    2016-01-01

    A series of hydroxy and phenolic compounds have been assayed for the inhibition of two physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II. The investigated molecules showed inhibition constants in the range of 1.07-4003 and 0.09-31.5 μM at the hCA I and hCA II enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are compared using three different scoring algorithms, namely Glide/SP, Glide/XP and Glide/IFD. In addition, different ADME (absorption, distribution, metabolism and excretion) analysis was performed. All the examined compounds were found within the acceptable range of pharmacokinetic profiles.

  16. Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors.

    PubMed

    Winum, Jean-Yves; Supuran, Claudiu T

    2015-04-01

    In addition to the sulfonamides and their isosteres, recently novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) which act by binding to the metal ion from the active site were discovered. Based on the X-ray crystal structure of the CA II-trithiocarbonate adduct, dithiocarbamates, xanthates and thioxanthates were shown to potently inhibit α- and β-CAs. The hydroxamates constitute another class of recently studied CAIs both against mammalian and protozoan enzymes. Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates, whereas the xanthates and salicylaldoximes were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates showed potent antiglaucoma activity in animal models of the disease whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA. PMID:24939097

  17. Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases from Cryptococcus neoformans, Candida albicans and Candida glabrata.

    PubMed

    Monti, Simona Maria; Maresca, Alfonso; Viparelli, Francesca; Carta, Fabrizio; De Simone, Giuseppina; Mühlschlegel, Fritz A; Scozzafava, Andrea; Supuran, Claudiu T

    2012-01-15

    A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the fungal pathogens Cryptococcus neoformans, Candida albicans and Candida glabrata, that is, Can2, CaNce103 and CgNce103, respectively. These enzymes were inhibited with efficacies between the subnanomolar to the micromolar range, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. This new class of β-CA inhibitors may have the potential for developing antifungal agents with a diverse mechanism of action compared to the clinically used drugs for which drug resistance was reported, and may also explain the efficacy of dithiocarbamates as agricultural antifungal agents. PMID:22209456

  18. Size and surface chemistry of nanoparticles lead to a variant behavior in the unfolding dynamics of human carbonic anhydrase

    NASA Astrophysics Data System (ADS)

    Nasir, Irem; Lundqvist, Martin; Cabaleiro-Lago, Celia

    2015-10-01

    The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence revealed that HCAs adsorb to both hydrophilic and hydrophobic surfaces, however the dynamics of the unfolding at the nanoparticle surfaces drastically vary with the polarity. The size of the nanoparticles has opposite effects depending on the polarity of the nanoparticle surface. The apolar nanoparticles induce seconds timescale structural rearrangements whereas polar nanoparticles induce hours timescale structural rearrangements on the same charged HCA variant. Here, a simple model is proposed where the difference in the timescales of adsorption is correlated with the energy barriers for initial docking and structural rearrangements which are firmly regulated by the surface polarity. Near-UV circular dichorism (CD) further supports that both protein variants undergo structural rearrangements at the nanoparticle surfaces regardless of being ``hard'' or ``soft''. However, the conformational changes induced by the apolar surfaces differ for each HCA isoform and diverge from the previously reported effect of silica nanoparticles.The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino

  19. X-ray crystallographic and kinetic investigations of 6-sulfamoyl-saccharin as a carbonic anhydrase inhibitor.

    PubMed

    Alterio, V; Tanc, M; Ivanova, J; Zalubovskis, R; Vozny, I; Monti, S M; Di Fiore, A; De Simone, G; Supuran, C T

    2015-04-01

    6-Sulfamoyl-saccharin was investigated as an inhibitor of 11 α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, hCA I-XIV, and X-ray crystallographic data were obtained for its adduct with hCA II, the physiologically dominant isoform. This compound possesses two potential zinc-binding groups, the primary sulfamoyl one and the secondary, acylatedsulfonamide. Saccharin itself binds to the Zn(II) ion from the CA active site coordinating with this last group, in deprotonated (SO2N(-)CO) form. Here we explain why 6-sulfamoyl-saccharin, unlike saccharin, binds to the metal ion from the hCA II active site by its primary sulfonamide moiety and not the secondary one as saccharin itself. Our study is useful for shedding new light to the structure-based drug design of isoform-selective CA inhibitors of the sulfonamide type.

  20. In vitro inhibition effect and structure-activity relationships of some saccharin derivatives on erythrocyte carbonic anhydrase I and II.

    PubMed

    Sonmez, Fatih; Bilen, Cigdem; Sumersan, Sinem; Gencer, Nahit; Isik, Semra; Arslan, Oktay; Kucukislamoglu, Mustafa

    2014-02-01

    In this study, in vitro inhibitory effects of some saccharin derivatives on purified carbonic anhydrase I and II were investigated using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among the compounds, 6-(p-tolylthiourenyl) saccharin (6m) was found to be the most active one for hCA I activity (IC50=13.67 μM) and 6-(m-methoxyphenylurenyl) saccharin (6b) was found to be the most active one for hCA II activity (IC50=6.54 μM). Structure-activity relationships (SARs) study showed that, generally, thiourea derivatives (6l--v) inhibited more hCA I and hCA II than urea derivatives (6a-k). All compounds (excluding 6c and 6r) have higher inhibitory activity on hCA II than on hCA I.

  1. Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

    PubMed Central

    Jamali, Somayeh; Klier, Michael; Ames, Samantha; Felipe Barros, L.; McKenna, Robert; Deitmer, Joachim W.; Becker, Holger M.

    2015-01-01

    The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+-shuttle His200, functions as a “proton-collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions. PMID:26337752

  2. Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

    PubMed

    Papalia, Giuseppe A; Leavitt, Stephanie; Bynum, Maggie A; Katsamba, Phinikoula S; Wilton, Rosemarie; Qiu, Huawei; Steukers, Mieke; Wang, Siming; Bindu, Lakshman; Phogat, Sanjay; Giannetti, Anthony M; Ryan, Thomas E; Pudlak, Victoria A; Matusiewicz, Katarzyna; Michelson, Klaus M; Nowakowski, Agnes; Pham-Baginski, Anh; Brooks, Jonathan; Tieman, Bryan C; Bruce, Barry D; Vaughn, Michael; Baksh, Michael; Cho, Yun Hee; Wit, Mieke De; Smets, Alexandra; Vandersmissen, Johan; Michiels, Lieve; Myszka, David G

    2006-12-01

    In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.

  3. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

  4. Molecular cloning, characterization, and inhibition studies of a β-carbonic anhydrase from Malassezia globosa, a potential antidandruff target.

    PubMed

    Hewitson, Kirsty S; Vullo, Daniela; Scozzafava, Andrea; Mastrolorenzo, Antonio; Supuran, Claudiu T

    2012-04-12

    A β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Malassezia globosa has been cloned, characterized, and studied for its inhibition with sulfonamides. This enzyme, designated MG-CA, has significant catalytic activity in the CO(2) hydration reaction and was inhibited by sulfonamides, sulfamates, and sulfamides with K(I) in the nanomolar to micromolar range. Several sulfonamides have also been investigated for the inhibition of growth of M. globosa, M. dermatis, M. pachydermatic, and M. furfur in cultures, whereas a mouse model of dandruff showed that treatment with sulfonamides led to fragmented fungal hyphae, as for the treatment with ketoconazole, a clinically used antifungal agent. These data prompt us to propose MG-CA as a new antidandruff drug target. PMID:22424239

  5. Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum

    PubMed Central

    Hirasawa, Michiru; Xu, Xinjie; Trask, Robert B.; Maddatu, Terry P.; Johnson, Britt A; Naggert, Jürgen K.; Nishina, Patsy M.; Ikeda, Akihiro

    2007-01-01

    Carbonic anhydrase related protein 8 (Car8) is known to be abundantly expressed in Purkinje cells (PCs), and its genetic mutation causes a motor coordination defect. To determine the underlying mechanism, we analyzed the mouse cerebellum carrying a Car8 mutation. Electrophysiological analysis showed that spontaneous excitatory transmission was largely diminished while paired pulse ratio at parallel fiber-PC synapses was comparable to wild-type, suggesting functional synapses have normal release probability but the number of functional synapses may be lower in mutants. Light microscopic study revealed an abnormal extension of climbing fibers to the distal PC dendrites. At ultrastructural level, we found numerous PC spines not forming synapses primarily in distal dendrites and occasionally multiple spines contacting a single varicosity. These abnormalities of parallel fiber-PC synapses may underlie the functional defect in excitatory transmission. Thus, Car8 plays a critical role in synaptogenesis and/or maintenance of proper synaptic morphology and function in the cerebellum. PMID:17376701

  6. Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase.

    PubMed

    Borén, Kristina; Grankvist, Hannah; Hammarström, Per; Carlsson, Uno

    2004-05-21

    During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.

  7. Significance of different carbon forms and carbonic anhydrase activity in monitoring and prediction of algal blooms in the urban section of Jialing River, Chongqing, China.

    PubMed

    Nie, Yudong; Zhang, Zhi; Shen, Qian; Gao, Wenjin; Li, Yingfan

    2016-05-18

    The Three Gorges Dam is one of the largest hydroelectric power plants worldwide; its reservoir was preliminarily impounded in 2003 and finally impounded to 175 m in 2012. The impoundment caused some environmental problems, such as algal blooms. Carbonic anhydrase (CA) is an important biocatalyst in the carbon utilization by algae and plays an important role in algal blooms. CA has received considerable attention for its role in red tides in oceans, but less investigation has been focused on its role in algal blooms in fresh water. In this study, the seasonal variation of water quality parameters, different carbon forms, carbonic anhydrase activity (CAA), and the algal cell density of four sampling sites in the urban section of the Jialing River were investigated from November 1, 2013 to October 31, 2014. Results indicated that CAA exhibited a positive correlation with dissoluble organic carbon (DOC), pH, and temperature, but a negative correlation with CO2 and dissoluble inorganic carbon (DIC). Algal cell density exhibited a positive correlation with flow velocity (V), pH, particulate organic carbon (POC), and CAA, a negative correlation with CO2, and a negative partial correlation with DIC. The relationship between CAA and algal cell density for the entire year can be described as cells = 23.278CAA - 42.666POC + 139.547pH - 1057.106. The algal bloom prediction model for the key control period can be described as cells = -45.895CAA + 776.103V- 29.523DOC + 14.219PIC + 35.060POC + 19.181 (2 weeks in advance) and cells = 69.200CAA + 203.213V + 4.184CO2 + 38.911DOC + 40.770POC - 189.567 (4 weeks in advance). The findings in this study demonstrate that the carbon utilization by algae is conducted by CA and provide a new method of monitoring algal cell density and predicting algal blooms.

  8. Significance of different carbon forms and carbonic anhydrase activity in monitoring and prediction of algal blooms in the urban section of Jialing River, Chongqing, China.

    PubMed

    Nie, Yudong; Zhang, Zhi; Shen, Qian; Gao, Wenjin; Li, Yingfan

    2016-05-18

    The Three Gorges Dam is one of the largest hydroelectric power plants worldwide; its reservoir was preliminarily impounded in 2003 and finally impounded to 175 m in 2012. The impoundment caused some environmental problems, such as algal blooms. Carbonic anhydrase (CA) is an important biocatalyst in the carbon utilization by algae and plays an important role in algal blooms. CA has received considerable attention for its role in red tides in oceans, but less investigation has been focused on its role in algal blooms in fresh water. In this study, the seasonal variation of water quality parameters, different carbon forms, carbonic anhydrase activity (CAA), and the algal cell density of four sampling sites in the urban section of the Jialing River were investigated from November 1, 2013 to October 31, 2014. Results indicated that CAA exhibited a positive correlation with dissoluble organic carbon (DOC), pH, and temperature, but a negative correlation with CO2 and dissoluble inorganic carbon (DIC). Algal cell density exhibited a positive correlation with flow velocity (V), pH, particulate organic carbon (POC), and CAA, a negative correlation with CO2, and a negative partial correlation with DIC. The relationship between CAA and algal cell density for the entire year can be described as cells = 23.278CAA - 42.666POC + 139.547pH - 1057.106. The algal bloom prediction model for the key control period can be described as cells = -45.895CAA + 776.103V- 29.523DOC + 14.219PIC + 35.060POC + 19.181 (2 weeks in advance) and cells = 69.200CAA + 203.213V + 4.184CO2 + 38.911DOC + 40.770POC - 189.567 (4 weeks in advance). The findings in this study demonstrate that the carbon utilization by algae is conducted by CA and provide a new method of monitoring algal cell density and predicting algal blooms. PMID:27142237

  9. The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability.

    PubMed

    James, Paul; Isupov, Michail N; Sayer, Christopher; Saneei, Vahid; Berg, Svein; Lioliou, Maria; Kotlar, Hans Kristian; Littlechild, Jennifer A

    2014-10-01

    Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold. PMID:25286845

  10. Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation.

    PubMed

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B; Simonsen, Ulf; Roepstorff, Andreas; Fago, Angela

    2009-12-01

    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 the reaction induces vasodilation in aortic rings. This reaction occurs under normoxic and hypoxic conditions and in various tissues at physiological levels of CA and nitrite. Furthermore, two specific inhibitors of the CO2 hydration, dorzolamide and acetazolamide, increase the CA-catalyzed production of vasoactive NO from nitrite. This enhancing effect may explain the known vasodilating effects of these drugs and indicates that CO2 and nitrite bind differently to the enzyme active site. Kinetic analyses show a higher reaction rate at high pH, suggesting that anionic nitrite participates more 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 blood flow and metabolic activity in tissues, as occurring for instance in active areas of the brain. PMID:19820197

  11. Activity and stability of immobilized carbonic anhydrase for promoting CO2 absorption into a carbonate solution for post-combustion CO2 capture.

    PubMed

    Zhang, Shihan; Zhang, Zhaohui; Lu, Yongqi; Rostam-Abadi, Massoud; Jones, Andrew

    2011-11-01

    An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90 days at 50 °C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4 M), nitrate (0.05 M) and chloride (0.3 M) typically found in flue gas scrubbing liquids than their free counterparts.

  12. Activity and stability of immobilized carbonic anhydrase for promoting CO2 absorption into a carbonate solution for post-combustion CO2 capture

    USGS Publications Warehouse

    Zhang, S.; Zhang, Z.; Lu, Y.; Rostam-Abadi, M.; Jones, A.

    2011-01-01

    An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90days at 50??C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4M), nitrate (0.05M) and chloride (0.3M) typically found in flue gas scrubbing liquids than their free counterparts. ?? 2011 Elsevier Ltd.

  13. Common Genetic Denominators for Ca++-Based Skeleton in Metazoa: Role of Osteoclast-Stimulating Factor and of Carbonic Anhydrase in a Calcareous Sponge

    PubMed Central

    Müller, Werner E. G.; Wang, Xiaohong; Grebenjuk, Vlad A.; Korzhev, Michael; Wiens, Matthias; Schloßmacher, Ute; Schröder, Heinz C.

    2012-01-01

    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 CaCl2) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca2+-depletion condition (1 mM CaCl2). 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). PMID:22506035

  14. Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis.

    PubMed

    Hofmann, Laurie C; Straub, Sandra; Bischof, Kai

    2013-02-01

    The concentration of CO(2) in global surface ocean waters is increasing due to rising atmospheric CO(2) emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO(2) concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO(2) concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO(2) concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO(2) and was highest in algae grown at 665 µatm CO(2). Nitrate and phosphate uptake rates were inversely related to CO(2), while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO(2). The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO(2) due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO(2) are discussed.

  15. Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis.

    PubMed

    Hofmann, Laurie C; Straub, Sandra; Bischof, Kai

    2013-02-01

    The concentration of CO(2) in global surface ocean waters is increasing due to rising atmospheric CO(2) emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO(2) concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO(2) concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO(2) concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO(2) and was highest in algae grown at 665 µatm CO(2). Nitrate and phosphate uptake rates were inversely related to CO(2), while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO(2). The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO(2) due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO(2) are discussed. PMID:23314813

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

    PubMed

    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.

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

    PubMed

    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

  18. 1. alpha. ,25-dihydroxyvitamin D sub 3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells

    SciTech Connect

    Billecocq, A.; Emanuel, J.R.; Levenson, R.; Baron, R. )

    1990-08-01

    1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25(OH){sub 2}D{sub 3}), the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH){sub 2}D{sub 3} could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH){sub 2}D{sub 3} in a dose-and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH){sub 2}D{sub 3} was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH){sub 2}D{sub 3} regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.

  19. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2014-06-01

    Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families.

  20. Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum

    PubMed Central

    Kikutani, Sae; Nakajima, Kensuke; Nagasato, Chikako; Tsuji, Yoshinori; Miyatake, Ai; Matsuda, Yusuke

    2016-01-01

    The algal pyrenoid is a large plastid body, where the majority of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO2-concentrating mechanism (CCM) and CO2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum. The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids. PMID:27531955

  1. An Overview of the Carbonic Anhydrases from Two Pathogens of the Oral Cavity: Streptococcus mutans and Porphyromonas gingivalis.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2016-01-01

    Among the crowd of bacteria provoking disease of the oral cavity during the weakened of immune system, Streptococcus mutans and Porphyromonas gingivalis are the main microorganisms implicated in caries formation and periodontitis, respectively. The life cycle of the pathogens, such as protozoa, fungi and bacteria, is influenced by a superfamily of enzymes, called carbonic anhydrases (CAs, EC 4.2.1.1). These metalloenzymes, being crucial for the survival of the pathogen, have been considered as novel anti-infective targets. In fact, bicarbonate and protons, produced by the CA catalyzed carbon dioxide as substrate, are two fundamental ions implicated in the pH regulation, biosynthetic reactions, and adaptation of the pathogen to the host or in the possibility of the pathogen to avoid the host immune system. Bacteria genome encodes for the α-, β- and γ-CAs. Recently, our groups using the recombinant DNA technology prepared and characterized the CAs belonging to the β- and γ-classes encoded by the genome of the two oral cavity pathogens S. mutans and P. gingivalis. An extensive inhibition study was carried out using typical anion/sulfonamide inhibitors of these classes of CAs. We discovered numerous inhibitors, which had in vitro an effective inhibitory activity against the bacterial CAs considered, here, as alternative anti-infective targets.

  2. Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.

    PubMed

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

    2016-12-01

    A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes. PMID:26497870

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

    PubMed

    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.

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

    PubMed

    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

  5. Identification of two carbonic anhydrases in the mantle of the European Abalone Haliotis tuberculata (Gastropoda, Haliotidae): phylogenetic implications.

    PubMed

    LE Roy, Nathalie; Marie, Benjamin; Gaume, Béatrice; Guichard, Nathalie; Delgado, Sidney; Zanella-Cléon, Isabelle; Becchi, Michel; Auzoux-Bordenave, Stéphanie; Sire, Jean-Yves; Marin, Frédéric

    2012-07-01

    Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks.

  6. Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum.

    PubMed

    Kikutani, Sae; Nakajima, Kensuke; Nagasato, Chikako; Tsuji, Yoshinori; Miyatake, Ai; Matsuda, Yusuke

    2016-08-30

    The algal pyrenoid is a large plastid body, where the majority of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO2-concentrating mechanism (CCM) and CO2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids. PMID:27531955

  7. Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase

    SciTech Connect

    J Mecinovic; P Snyder; K Mirica; S Bai; E Mack; R Kwant; D Moustakas; A Heroux; G Whitesides

    2011-12-31

    The hydrophobic effect, the free-energetically favorable association of nonpolar solutes in water, makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different but structurally similar hydrophobic groups, aliphatic hydrocarbons and aliphatic fluorocarbons, and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H{sub 2}NSO{sub 2}C{sub 6}H{sub 4}-CONHCH{sub 2}(CX{sub 2}){sub n}CX{sub 3}, n = 0-4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of nonoptimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and

  8. Effects of ocean acidification on the photosynthetic performance, carbonic anhydrase activity and growth of the giant kelp Macrocystis pyrifera.

    PubMed

    Fernández, Pamela A; Roleda, Michael Y; Hurd, Catriona L

    2015-06-01

    Under ocean acidification (OA), the 200 % increase in CO2(aq) and the reduction of pH by 0.3-0.4 units are predicted to affect the carbon physiology and growth of macroalgae. Here we examined how the physiology of the giant kelp Macrocystis pyrifera is affected by elevated pCO2/low pH. Growth and photosynthetic rates, external and internal carbonic anhydrase (CA) activity, HCO3 (-) versus CO2 use were determined over a 7-day incubation at ambient pCO2 400 µatm/pH 8.00 and a future OA treatment of pCO2 1200 µatm/pH 7.59. Neither the photosynthetic nor growth rates were changed by elevated CO2 supply in the OA treatment. These results were explained by the greater use of HCO3 (-) compared to CO2 as an inorganic carbon (Ci) source to support photosynthesis. Macrocystis is a mixed HCO3 (-) and CO2 user that exhibits two effective mechanisms for HCO3 (-) utilization; as predicted for species that possess carbon-concentrating mechanisms (CCMs), photosynthesis was not substantially affected by elevated pCO2. The internal CA activity was also unaffected by OA, and it remained high and active throughout the experiment; this suggests that HCO3 (-) uptake via an anion exchange protein was not affected by OA. Our results suggest that photosynthetic Ci uptake and growth of Macrocystis will not be affected by elevated pCO2/low pH predicted for the future, but the combined effects with other environmental factors like temperature and nutrient availability could change the physiological response of Macrocystis to OA. Therefore, further studies will be important to elucidate how this species might respond to the global environmental change predicted for the ocean.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions. PMID:25865133

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

    PubMed

    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

  12. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions.

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

    PubMed Central

    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 to small cracks formed at early age of 7 days, cracks below 0.4 mm was almost completely closed. The repair effect reduced with the increasing of cracking age. Cracks width influenced self-healing effectiveness significantly. The transportation of CO2and Ca2+ controlled the self-healing process. The computer simulation analyses revealed the self-healing process and mechanism of microbiologically precipitation induced by bacteria and the depth of precipitated CaCO3 could be predicted base on valid Ca2+. PMID:26583014

  14. Hydrophobic Substituents of the Phenylmethylsulfamide Moiety Can Be Used for the Development of New Selective Carbonic Anhydrase Inhibitors

    PubMed Central

    Pizika, Ginta; Monti, Simona Maria; Di Fiore, Anna; Trapencieris, Peteris; Supuran, Claudiu T.; Alterio, Vincenzo

    2014-01-01

    A new series of compounds containing a sulfamide moiety as zinc-binding group (ZBG) has been synthesized and tested for determining inhibitory properties against four human carbonic anhydrase (hCA) isoforms, namely, CAs I, II, IX, and XII. The X-ray structure of the cytosolic dominant isoform hCA II in complex with the best inhibitor of the series has also been determined providing further insights into sulfamide binding mechanism and confirming that such zinc-binding group, if opportunely derivatized, can be usefully exploited for obtaining new potent and selective CAIs. The analysis of the structure also suggests that for drug design purposes the but-2-yn-1-yloxy moiety tail emerges as a very interesting substituent of the phenylmethylsulfamide moiety due to its capability to establish strong van der Waals interactions with a hydrophobic cleft on the hCA II surface, delimited by residues Phe131, Val135, Pro202, and Leu204. Indeed, the complementarity of this tail with the cleft suggests that different substituents could be used to discriminate between isoforms having clefts with different sizes. PMID:25258712

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

    PubMed Central

    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

  16. Inhibition studies of new ureido-substituted sulfonamides incorporating a GABA moiety against human carbonic anhydrase isoforms I-XIV.

    PubMed

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

    2014-12-15

    Reaction of γ-Boc-GABA, prepared by protecting the γ-amino moiety of the amino butyric acid with the tert-butyloxycarbonyl (Boc) protecting group, with 4-methyl/ethyl benzenesulfonamide, followed by removal of the Boc protecting group in 3 M HCl afforded the corresponding hydrochlorides, which were further derivatized by reaction with a varying of aryl isocyanates to give a new classes of ureido substituted benzenesulfonamide containing a GABA moiety. Inhibition studies of the human carbonic anhydrase(CA, EC 4.2.1.1) isoforms, CA I–XIV with these new compounds revealed that they possess moderate-weak inhibition potency against hCA III, IV, VA, VI and XIII, rather efficient inhibitory power against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II and VII, as well as of the two tumor-associated isoforms CA IX and XII. The inhibition profile of the new ureido-substituted benzenesulfonamides reported here is thus very different from the corresponding ureido-substituted analogs incorporating sulfanilamide, which were previously investigated as inhibitors of some of these enzymes. PMID:25468040

  17. Sulfonamide inhibition study of the β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans.

    PubMed

    Dedeoglu, Nurcan; DeLuca, Viviana; Isik, Semra; Yildirim, Hatice; Kockar, Feray; Capasso, Clemente; Supuran, Claudiu T

    2015-06-01

    Streptococcus mutans, the oral pathogenic bacterium provoking dental caries formation, encodes for a β-class carbonic anhydrase (CA, EC 4.2.1.1), SmuCA. This enzyme was cloned, characterized and investigated for its inhibition profile with the major class of CA inhibitors, the primary sulfonamides. SmuCA has a good catalytic activity for the CO2 hydration reaction, with a kcat of 4.2×10(5) s(-1) and kcat/Km of 5.8×10(7) M(-1)×s(-1), and is efficiently inhibited by most sulfonamides (KIs of 246 nM-13.5 μM). The best SmuCA inhibitors were bromosulfanilamide, deacetylated acetazolamide, 4-hydroxymethylbenzenesulfonamide, a pyrimidine-substituted sulfanilamide derivative, aminobenzolamide and compounds structurally similar to it, as well as acetazolamide, methazolamide, indisulam and valdecoxib. These compounds showed inhibition constants ranging between 246 and 468 nM. Identification of effective inhibitors of this enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action. PMID:25913199

  18. Characterization and inhibition studies of an α-carbonic anhydrase from the endangered sturgeon species Acipenser gueldenstaedti.

    PubMed

    Kolayli, Sevgi; Karahalil, Fatma; Sahin, Huseyin; Dincer, Barbaros; Supuran, Claudiu T

    2011-12-01

    An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from erythrocytes of the sturgeon Acipenser gueldenstaedti, an endangered species. The sturgeon enzyme (AgCA) showed kinetic parameters for the CO(2) hydration reaction comparable with those of the human erythrocytes enzyme hCA II, being a highly active enzyme, whereas its esterase activity with 4-nitrophenyl acetate as substrate was lower. Sulphonamide inhibitors (acetazolamide, sulphanilamide) strongly inhibited AgCA, whereas metal ions (Ag(+), Zn(2+), Cu(2+) and Co(2+)) were weak, millimolar inhibitors. Several widely used pesticides (2,4-dichlorophenol, dithiocarbamates, parathion and carbaryl) were also assayed as inhibitors of this enzyme. The dithiocarbamates were low micromolar AgCA inhibitors (IC(50) of 16-18 μM), whereas the other pesticides inhibited the enzyme with IC(50)s in the range of 102-398 μM. The wide use of dithiocarbamate pesticides may be one of the factors enhancing the vulnerability of this sturgeon species to pollutants. PMID:21381885

  19. Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties.

    PubMed

    Ceruso, Mariangela; Carta, Fabrizio; Osman, Sameh M; Alothman, Zeid; Monti, Simona Maria; Supuran, Claudiu T

    2015-08-01

    A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three β-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high β-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs. PMID:26145821

  20. Characterization and anions inhibition studies of an α-carbonic anhydrase from the teleost fish Dicentrarchus labrax.

    PubMed

    Ekinci, Deniz; Ceyhun, Saltuk Buğrahan; Sentürk, Murat; Erdem, Deryanur; Küfrevioğlu, Omer İrfan; Supuran, Claudiu T

    2011-01-15

    Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a specific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10°C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H₂NSO₃⁻, I⁻, SCN⁻, NO₃⁻, NO₂⁻, N₃⁻, Br⁻, Cl⁻, SO₄²⁻, and F⁻ showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K(i)s of 87-187 μM). NO₃⁻, NO₂⁻ and N₃⁻ were moderate inhibitors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a competitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the fish and mammalian CAs. PMID:21211980

  1. Dihalogenated sulfanilamides and benzolamides are effective inhibitors of the three β-class carbonic anhydrases from Mycobacterium tuberculosis.

    PubMed

    Maresca, Alfonso; Scozzafava, Andrea; Vullo, Daniela; Supuran, Claudiu T

    2013-04-01

    A series of halogenated sulfanilamides and halogenated benzolamide derivatives have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the substitution pattern at the sulfanilamide moiety/fragment of the molecule. Best inhibitors were the halogenated benzolamides (K(I)s in the range of 0.12-0.45 μM) whereas the halogenated sulfanilamides were slightly less inhibitory (K(I)s in the range of 0.41-4.74 μM). This class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance. PMID:22214209

  2. High expression and biosilica encapsulation of alkaline-active carbonic anhydrase for CO2 sequestration system development.

    PubMed

    Min, Ki-Ha; Son, Ryeo Gang; Ki, Mi-Ran; Choi, Yoo Seong; Pack, Seung Pil

    2016-01-01

    Carbonic anhydrase (CA) is a biocatalyst for CO2 sequestration because of its distinctive ability to accelerate CO2 hydration. High production and efficient immobilization of alkaline-active CAs are required, because one potential application of CA is its use in the alkaline solvent-based CO2 absorption/desorption process. Here, we designed and applied an α-type CA from Hahella chejuensis (HCA), which was reported as highly active in alkaline conditions, but was mostly expressed as insoluble forms. We found that the signal peptide-removed form of HCA [HCA(SP-)] was successfully expressed in the soluble form [∼70mg of purified HCA(SP-) per L of culture]. HCA(SP-) also displayed high pH stability in alkaline conditions, with maximal activity at pH 10; at this pH, ∼90% activity was maintained for 2h. Then, we prepared HCA(SP-)-encapsulated silica particles [HCA(SP-)@silica] via a spermine-mediated bio-inspired silicification method. HCA(SP-)@silica exhibited high-loading and highly stable CA activity. In addition, HCA(SP-)@silica retained more than 90% of the CA activity even after 10 cycles of use in mild conditions, and ∼80% in pH 10 conditions. These results will be useful for the development of practical CO2 sequestration processes employing CA.

  3. DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

    PubMed Central

    Sung, Hye Youn

    2014-01-01

    Purpose Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. Materials and Methods We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. Results Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. Conclusion Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells. PMID:25323905

  4. Cryoannealing-induced space-group transition of crystals of the carbonic anhydrase psCA3.

    PubMed

    Pinard, Melissa A; Kurian, Justin J; Aggarwal, Mayank; Agbandje-McKenna, Mavis; McKenna, Robert

    2016-07-01

    Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the β-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P21212 and was scaled to 2.6 Å resolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Å in resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor. PMID:27380376

  5. Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties.

    PubMed

    Ceruso, Mariangela; Carta, Fabrizio; Osman, Sameh M; Alothman, Zeid; Monti, Simona Maria; Supuran, Claudiu T

    2015-08-01

    A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three β-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high β-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.

  6. Anion inhibition studies of the dandruff-producing fungus Malassezia globosa β-carbonic anhydrase MgCA.

    PubMed

    Del Prete, Sonia; Vullo, Daniela; Osman, Sameh M; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2015-11-15

    The genome of the fungal parasite Malassezia globosa, the causative agent of dandruff, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). In an earlier work (J. Med. Chem. 2012, 55, 3513) we have validated this enzyme as an anti-dandruff drug target, reporting that sulfonamide inhibitors show in vitro and in vivo effects, in an animal model of Malassezia infection. However, few classes of compounds apart the sulfonamides, were investigated for their activity against MgCA. Here we present an anion inhibition study of this enzyme, reporting that metal complexing anions such as cyanate, thiocyanate, cyanide, azide are weak MgCA inhibitors (KIs ranging between 6.81 and 45.2 mM) whereas bicarbonate (KI of 0.59 mM) and diethyldithiocarbamate (KI of 0.30 mM) together with sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected so far, with KIs ranging between 83 and 94 μM. This study may help a better understanding of the inhibition profile of this enzyme and may offer the possibility to design new such modulators of activity belonging to different chemical classes. PMID:26459213

  7. Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models

    PubMed Central

    Chen, Liu Qi; Howison, Christine M.; Spier, Catherine; Stopeck, Alison T.; Malm, Scott W.; Pagel, Mark D.; Baker, Amanda F.

    2015-01-01

    The expression of carbonic anhydrase (CA IX) and it’s relation to acidosis in lymphomas has not been widely studied. We investigated the protein expression of CA IX in a human B-cell lymphoma tissue microarray, and in Raji, Ramos, and Granta 519 lymphoma cell lines and tumor models, while also investigating the relation with hypoxia. An imaging method, acidoCEST MRI, was used to estimate lymphoma xenograft extracellular pH (pHe). Our results showed that clinical lymphoma tissues and cell line models in vitro and in vivo had moderate CA IX expression. Although in vitro studies showed that CA IX expression was induced by hypoxia, in vivo studies did not show this correlation. Untreated lymphoma xenograft tumor pHe had acidic fractions, and an Acidity Score was qualitatively correlated with CA IX expression. Therefore, CA IX is expressed in B-cell lymphomas and is qualitatively correlated with extracellular acidosis in xenograft tumor models. PMID:25130478

  8. Mitochondrial carbonic anhydrase VA deficiency resulting from CA5A alterations presents with hyperammonemia in early childhood.

    PubMed

    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

    2014-03-01

    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

  9. Synthesis and carbonic anhydrase inhibitory properties of amino acid - coumarin/quinolinone conjugates incorporating glycine, alanine and phenylalanine moieties.

    PubMed

    Küçükbay, F Zehra; Küçükbay, Hasan; Tanc, Muhammet; Supuran, Claudiu T

    2016-12-01

    N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and quinolinone derivatives, leading to the corresponding N-protected amino acid-coumarin/quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against various human (h) isoforms, such as hCA I, hCA II, hCA IV and hCA XII. The quinolinone conjugates were inactive as enzyme inhibitors, whereas the coumarins were ineffective hCA I/II inhibitors (KIs > 50 μM) but were submicromolar hCA IV and XII inhibitors, with inhibition constants ranging between 92 nM and 1.19 μM for hCA IV, and between 0.11 and 0.79 μM for hCA XII. These coumarin derivatives, as many others reported earlier, thus show an interesting selective inhibitory profile for the membrane-bound over the cytosolic CA isoforms.

  10. Transport Activity of the Sodium Bicarbonate Cotransporter NBCe1 Is Enhanced by Different Isoforms of Carbonic Anhydrase

    PubMed Central

    Schueler, Christina; Becker, Holger M.; McKenna, Robert; Deitmer, Joachim W.

    2011-01-01

    Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity. PMID:22076132

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

    PubMed

    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

  12. Carbonic anhydrase inhibitors: Design, synthesis, kinetic, docking and molecular dynamics analysis of novel glycine and phenylalanine sulfonamide derivatives.

    PubMed

    Fidan, İsmail; Salmas, Ramin Ekhteiari; Arslan, Mehmet; Şentürk, Murat; Durdagi, Serdar; Ekinci, Deniz; Şentürk, Esra; Coşgun, Sedat; Supuran, Claudiu T

    2015-12-01

    The inhibition of two human cytosolic carbonic anhydrase isozymes I and II, with some novel glycine and phenylalanine sulfonamide derivatives were investigated. Newly synthesized compounds G1-4 and P1-4 showed effective inhibition profiles with KI values in the range of 14.66-315μM for hCA I and of 18.31-143.8μM against hCA II, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico docking studies were applied. Atomistic molecular dynamic simulations were performed for docking poses which utilize to illustrate the inhibition mechanism of used inhibitors into active site of CAII. These sulfonamide containing compounds generally were competitive inhibitors with 4-nitrophenylacetate as substrate. Some investigated compounds here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide, sulfanilamide or mafenide and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents. PMID:26534780

  13. Expression of testosterone-dependent enzyme, carbonic anhydrase III, and oxidative stress in experimental alcoholic liver disease.

    PubMed

    Parkkila, S; Halsted, C H; Villanueva, J A; Väänänen, H K; Niemelä, O

    1999-11-01

    We studied the sequential immunohistochemical appearance of androgen-dependent carbonic anhydrase (CA III) during the development of ethanol-induced liver injury using liver samples from castrated and noncastrated male micropigs. In castrated micropigs, the baseline expression of CA III was either low or absent, while distinct positive immunoreactions were found in zone 3 hepatocytes at 5 and 12 months after the initiation of the ethanol diet. The CA III enzyme and protein adducts of lipid peroxidation-derived aldehydic products, malondialdehyde and 4-hydroxynonenal, appeared together in the perivenous region, suggesting that the enzyme functions in an oxidative environment. The positive staining became more abundant and widespread during the progression of alcoholic liver disease. After 12 months, CA III was significantly more abundant in both the ethanol-fed noncastrated and castrated micropigs than in the control animals (P < 0.001, P < 0.05, respectively). CA III content was strikingly high in the ethanol-fed noncastrated animals, consistent with a potential role of androgens in the regulation of ethanol-induced CA III expression. The strongly positive CA III immunoreactions in the ethanol-fed noncastrated micropigs were associated with scant evidence of aldehydic protein adducts and minimal histopathology. Thus, enhanced expression of CA III during ethanol consumption may also account in part for gender differences in the susceptibility for alcohol-induced liver injury.

  14. Intrinsic thermodynamics of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding to carbonic anhydrases by isothermal titration calorimetry.

    PubMed

    Zubrienė, Asta; Smirnovienė, Joana; Smirnov, Alexey; Morkūnaitė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Juozapaitienė, Vaida; Norvaišas, Povilas; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

    2015-10-01

    Para substituted tetrafluorobenzenesulfonamides bind to carbonic anhydrases (CAs) extremely tightly and exhibit some of the strongest known protein-small ligand interactions, reaching an intrinsic affinity of 2 pM as determined by displacement isothermal titration calorimetry (ITC). The enthalpy and entropy of binding to five CA isoforms were measured by ITC in two buffers of different protonation enthalpies. The pKa values of compound sulfonamide groups were measured potentiometrically and spectrophotometrically, and enthalpies of protonation were measured by ITC in order to evaluate the proton linkage contributions to the observed binding thermodynamics. Intrinsic means the affinity of a sulfonamide anion for the Zn bound water form of CAs. Fluorination of the benzene ring significantly enhanced the observed affinities as it increased the fraction of deprotonated ligand while having little impact on intrinsic affinities. Intrinsic enthalpy contributions to the binding affinity were dominant over entropy and were more exothermic for CA I than for other CA isoforms. Thermodynamic measurements together with the X-ray crystallographic structures of protein-ligand complexes enabled analysis of structure-activity relationships in this enzyme ligand system.

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

    PubMed

    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.

  16. Exploring associations between taste perception, oral anatomy and polymorphisms in the carbonic anhydrase (gustin) gene CA6.

    PubMed

    Feeney, Emma L; Hayes, John E

    2014-04-10

    Recent reports suggest that polymorphisms in the carbonic anhydrase gene CA6 (also known as gustin) may explain additional variation in the bitterness of 6-n-propylthiouracil beyond that explained by variation in the bitter receptor gene TAS2R38. CA6 (gustin) has been implicated in taste bud function and salivary buffer capacity. In the present study we examined associations between polymorphisms in the CA6 gene with salt and bitter taste perception, and oral anatomy. 243 subjects (146 female) aged 18-45 rated the intensity of five concentrations of 6-n-propylthiouracil and NaCl on a generalized Labeled Magnitude Scale (gLMS) in duplicate and one concentration of potassium chloride (KCl). Using salivary DNA, we examined 12 SNPs within CA6 in relation to taste intensity and number of fungiform papillae. We observed no difference in bitter taste perception from 6-n-propylthiouracil (PROP) or from potassium chloride for any of the SNPs examined. Perceived saltiness of NaCl on the other hand was significantly associated with a number of CA6 polymorphisms, and particularly rs3737665. Nonetheless, FP density did not vary between alleles of rs3737665, nor with any of the other CA6 SNPs. Also, we fail to find any evidence that CA6 effects on taste perception are due to differences in fungiform papilla number. Additional work is needed to confirm whether variations within the CA6 gene may be responsible for differences in salt taste perception.

  17. Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II.

    PubMed

    Gökçe, Başak; Gençer, Nahit; Arslan, Oktay; Turkoğlu, Sumeyye Aydogan; Alper, Meltem; Köçkar, Feray

    2012-02-01

    The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC(50) values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC(50) concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 μM and 4250 μM and for hCA II 950 μM and 6200 μM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes. PMID:21534860

  18. Mitochondrial carbonic anhydrase VA deficiency resulting from CA5A alterations presents with hyperammonemia in early childhood.

    PubMed

    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

    2014-03-01

    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.

  19. Carbonic anhydrase inhibitors: Design, synthesis, kinetic, docking and molecular dynamics analysis of novel glycine and phenylalanine sulfonamide derivatives.

    PubMed

    Fidan, İsmail; Salmas, Ramin Ekhteiari; Arslan, Mehmet; Şentürk, Murat; Durdagi, Serdar; Ekinci, Deniz; Şentürk, Esra; Coşgun, Sedat; Supuran, Claudiu T

    2015-12-01

    The inhibition of two human cytosolic carbonic anhydrase isozymes I and II, with some novel glycine and phenylalanine sulfonamide derivatives were investigated. Newly synthesized compounds G1-4 and P1-4 showed effective inhibition profiles with KI values in the range of 14.66-315μM for hCA I and of 18.31-143.8μM against hCA II, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico docking studies were applied. Atomistic molecular dynamic simulations were performed for docking poses which utilize to illustrate the inhibition mechanism of used inhibitors into active site of CAII. These sulfonamide containing compounds generally were competitive inhibitors with 4-nitrophenylacetate as substrate. Some investigated compounds here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide, sulfanilamide or mafenide and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

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

    PubMed

    Muselaers, Constantijn H J; Oosterwijk, Egbert; Bos, Desirée L; Oyen, Wim J G; Mulders, Peter F A; Boerman, Otto 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 study in mice with intraperitoneally growing ccRCC lesions. Mice with intraperitoneal xenografts were injected with 1, 3, 10, 30, or 100 μg of G250 labeled with 10 MBq indium 111 (111In) to determine the optimal protein dose. The optimal protein dose determined with imaging and biodistribution studies was used in a subsequent RIT experiment in three groups of 10 mice with intraperitoneal SK-RC-52 tumors. One group received 13 MBq 177Lu-DOTA-G250, a control group received 13 MBq nonspecific 177Lu-MOPC21, and the second control group was not treated and received 20 MBq 111In-DOTA-G250. The optimal G250 protein dose to target ccRCC in this model was 10 μg G250. Treatment with 13 MBq 177Lu-DOTA-G250 was well tolerated and resulted in significantly prolonged median survival (139 days) compared to controls (49-53 days, p  =  .015), indicating that RIT has potential in this metastatic ccRCC model.

  1. A Class of 4-Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective Carbonic Anhydrase Inhibitory Action and Antiglaucoma Effects

    PubMed Central

    2015-01-01

    We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure–activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a–c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII. PMID:25358036

  2. Population genetic studies of the Philippine Negritos. III. Identification of the carbonic anhydrase-1 variant with CA1 Guam.

    PubMed Central

    Omoto, K; Ueda, S; Goriki, K; Takahashi, N; Misawa, S; Pagaran, I G

    1981-01-01

    Investigation of blood samples from 277 Mamanwas of northeastern Mindanao, Philippines, confirmed the concentration of the variant carbonic anhydrase-1 (CA1 3N) in this group. The frequency for the variant allele was estimated at .217 +/- .017. It occurs also in the Manobos, the Mongoloid indigenous inhabitants of the same district, although the frequency is low (.019 +/- .008). Survey of samples from other Philippine populations, including the Aeta and the Ifugao of Luzon, failed to find variants. This findings suggests different origins of the Aeta and the Mamanwa, although both are usually referred to as Negritos. The Ca1 3N protein was purified by affinity chromatography using azosulfonamide and rechromatography on a DEAE-Sephadex column. The tryptic peptide pattern of CA1 3N was similar to that of CA1 Guam already reported. Furthermore, amino acid analyses of the tryptic peptides indicated that CA1 3N is characterized by the substitution 253 Gly leads to Arg, confirming the identity of this variant with CA1 Guam. The widespread occurrence of CA1 3 variants in the Western Pacific suggests that this variant was once common in an aboriginal population of this region, from which it was scattered by gene flow. Images Fig. 1 Fig. 2 PMID:6781336

  3. Dexamethasone downregulates expression of carbonic anhydrase IX via HIF-1α and NF-κB-dependent mechanisms

    PubMed Central

    Simko, Veronika; Takacova, Martina; Debreova, Michaela; Laposova, Katarina; Ondriskova-Panisova, Elena; Pastorekova, Silvia; Csaderova, Lucia; Pastorek, Jaromir

    2016-01-01

    Dexamethasone is a synthetic glucocorticoid frequently used to suppress side-effects of anticancer chemotherapy. In the present study, we showed that dexamethasone treatment leads to concentration-dependent downregulation of cancer-associated marker, carbonic anhydrase IX (CA IX), at the level of promoter activity, mRNA and protein expression in 2D and 3D cancer cell models. The effect of dexamethasone on CA IX expression under hypoxic conditions is predominantly mediated by impaired transcriptional activity and decreased protein level of the main hypoxic transcription factor HIF-1α. In addition, CA9 downregulation can be caused by protein-protein interactions between activated glucocorticoid receptors, major effectors of glucocorticoid action, and transcription factors that trigger CA9 transcription (e.g. AP-1). Moreover, we identified a potential NF-κB binding site in the CA9 promoter and propose the involvement of NF-κB in the dexamethasone-mediated inhibition of CA9 transcription. As high level of CA IX is often linked to aggressive tumor behavior, poor prognosis and chemo- and radiotherapy resistance, uncovering its reduction after dexa-methasone treatment and implication of additional regulatory mechanisms can be relevant for the CA IX-related clinical applications. PMID:27431580

  4. Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: a one transcription factor (HIF-1) show?

    PubMed Central

    Kaluz, Stefan; Kaluzová, Milota; Liao, Shu-Yuan; Lerman, Michael; Stanbridge, Eric J

    2009-01-01

    SUMMARY Transcriptional activation by hypoxia is mediated by the hypoxia-inducible factor (HIF) via binding to the hypoxia-responsive element (HRE). Hypoxia in solid tumors associates with poorer outcome of the disease and reliable cellular markers of tumor hypoxia would represent a valuable diagnostic marker and a potential therapeutic target. In this category, carbonic anhydrase IX (CAIX) is one of the most promising candidates. Here, we summarize the knowledge about transcriptional regulation of CA9. The HRE is the central regulatory element in the CA9 promoter, whereas other elements are limited to lesser roles of amplification of signals received at the HRE. The analysis of known mechanisms of activation of CA9 reveals the prominent role of the HIF-1 pathway. Experimental paradigms with uncoupled HIF-1α stability and transcriptional activity (pericellular hypoxia, proteasomal inhibitor) provide evidence that CA9 expression monitors transcriptional activity of HIF-1, rather than the abundance of HIF-1α. Furthermore, these paradigms could provide a corollary to some of the apparently discordant cases (CAIX+, HIF-1α−) or (CAIX−, HIF-1α+) observed in vivo. In conclusion, the existing data support the notion that CA9, due to the unique structure of its promoter, is one of the most sensitive endogenous sensors of HIF-1 activity. PMID:19344680

  5. Carbonic anhydrase activators: an activation study of the human mitochondrial isoforms VA and VB with amino acids and amines.

    PubMed

    Vullo, Daniela; Nishimori, Isao; Innocenti, Alessio; Scozzafava, Andrea; Supuran, Claudiu T

    2007-03-01

    The mitochondrial isozymes of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VA and hCA VB, were investigated for activation with a series of amino acids and amines. D-His, L-DOPA, histamine, dopamine, and 4-(2-aminoethyl)morpholine were excellent hCA VA activators, with KAs in the range of 10-130 nM. Good hCA VB activating effects were identified for L-His, D-Phe, D-DOPA, L-Trp, L-Tyr, serotonin, and 2-(2-aminoethyl)-pyridine, with KAs in the range of 44-110 nM. All these activators enhanced kcat, having no effect on KM, favoring thus the rate-determining step in the catalytic cycle, the proton transfer reactions between the active site and environment. The activation pattern of the two mitochondrial isoforms is very different from each other and as compared to those of the cytosolic isoforms hCA I and II. PMID:17174092

  6. Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans.

    PubMed

    Schlicker, Christine; Hall, Rebecca A; Vullo, Daniela; Middelhaufe, Sabine; Gertz, Melanie; Supuran, Claudiu T; Mühlschlegel, Fritz A; Steegborn, Clemens

    2009-01-30

    In the pathogenic fungus Cryptococcus neoformans, a CO(2)-sensing system is essential for survival in the natural environment (approximately 0.03% CO(2)) and mediates the switch to virulent growth in the human host (approximately 5% CO(2)). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the "plant-type" beta-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors. PMID:19071134

  7. Inhibition of carbonic anhydrase isozymes I and II with natural products extracted from plants, mushrooms and honey.

    PubMed

    Sahin, Huseyin; Can, Zehra; Yildiz, Oktay; Kolayli, Sevgi; Innocenti, Alessio; Scozzafava, Gabriele; Supuran, Claudiu T

    2012-06-01

    Different natural products and secondary metabolites from mushrooms, teas, honeys, mosses, plants and seaweeds were investigated for their in vitro inhibitory effects on human carbonic anhydrase (hCA, E.C.4.2.1.1) isoforms I and II. Inhibition data were correlated with the total phenol content in the extract and investigated with the pure compounds believed to be responsible for this activity. Methanolic extracts were prepared for 17 such pure chemicals present in the natural products and for 41 diverse natural products. The IC(50) values were in the range of 0.11-66.50 μg/mL against hCA I and of 0.09-54.54 μg/mL against hCA II, respectively. The total phenol content was in the range of 0.02-1318.96 (as milligrams of gallic acid equivalents) per gram of sample. These data offer new insights on possible novel classes of CA inhibitors based on natural products, possessing a range of chemical structures not present in the classical inhibitors with pharmacological applications, such as the sulfonamides and sulfamates. PMID:21740099

  8. Cloning, characterization and anion inhibition studies of a new γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

    PubMed

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

    2015-08-01

    A new γ-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCAγ. The enzyme has a medium-low catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a kcat of 1.4×10(5)s(-1) and a kcat/Km of 1.9×10(6)M(-1)s(-1). An anion inhibition study of PhaCAγ with inorganic anions and small molecule inhibitors is also reported. Many anions present in sea water, such as chloride, fluoride, sulfate, iodide, but also others such as azide, perchlorate and tetrafluoroborate did not inhibit this enzyme. Pseudohalides such as cyanate, thiocyanate, cyanide, selenocyanide, and also bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range (KI in the range of 1.7-9.3mM). The best PhaCAγ inhibitors detected in this study were diethyldithiocarbamate (KI of 0.96 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KI in the range of 82-91 μM). Since γ-CAs are poorly understood at this moment, being present in carboxysomes and thus involved in photosynthesis, this study may be relevant for a better understanding of these processes in Antarctic bacteria/cyanobacteria. PMID:26145820

  9. Structural Studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa: Inhibitor Complexes with Anions and Acetazolamide

    PubMed Central

    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 CO2-HCO3- 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

  10. Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus.

    PubMed

    Calhoun, Stacy; Zou, Enmin

    2016-03-01

    During the crustacean molting cycle, the exoskeleton is first mineralized in postmolt and intermolt and then presumably demineralized in premolt in order for epidermal retraction to occur. The mineralization process calls for divalent metal ions, such as Ca(2+) and Mg(2+) , and bicarbonate ions whereas protons are necessary for dissolution of carbonate salts. Carbonic anhydrase (CA) has been suggested to be involved in exoskeletal mineralization by providing bicarbonate ions through catalyzing the reaction of carbon dioxide hydration. However, results of earlier studies on the role of epidermal CA in metal incorporation in crustacean exoskeleton are not consistent. This study was aimed to provide further evidence to support the notion that epidermal CA is involved in exoskeletal mineralization using the blue crab, Callinectes sapidus (Rathbun 1896), as the model crustacean. Significant increases first in calcium and magnesium then in manganese post-ecdysis indicate significant metal deposition during postmolt and intermolt. Significant positive correlation between calcium or magnesium content and epidermal CA activity in postmolt and intermolt constitutes evidence that CA is involved in the mineralization of the crustacean exoskeleton. Additionally, we proposed a hypothetical model to describe the role of epidermal CA in both mineralization and demineralization of the exoskeleton based on the results of epidermal CA activity and exoskeletal metal content during the molting cycle. Furthermore, we found that the pattern of epidermal CA activity during the molting cycle of C. sapidus is similar to that of ecdysteroids reported for the same species, suggesting that epidermal CA activity may be under control of the molting hormones. PMID:26935248

  11. Internal carbonic anhydrase activity in the tissue of scleractinian corals is sufficient to support proposed roles in photosynthesis and calcification.

    PubMed

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

    2015-07-01

    Reef-building corals import inorganic carbon (Ci) to build their calcium carbonate skeletons and to support photosynthesis by the symbiotic algae that reside in their tissue. The internal pathways that deliver Ci for both photosynthesis and calcification are known to involve the enzyme carbonic anhydrase (CA), which interconverts CO2 and HCO3 (-). We have developed a method for absolute quantification of internal CA (iCA) activity in coral tissue based on the rate of (18)O-removal from labeled Ci. The method was applied to three Caribbean corals (Orbicella faveolata, Porites astreoides and Siderastrea radians) and showed that these species have similar iCA activities per unit surface area, but that S. radians has ∼10-fold higher iCA activity per unit tissue volume. A model of coral Ci processing shows that the measured iCA activity is sufficient to support the proposed roles for iCA in Ci transport for photosynthesis and calcification. This is the case even when iCA activity is homogeneously distributed throughout the coral, but the model indicates that it would be advantageous to concentrate iCA in the spaces where calcification (the calcifying fluid) and photosynthesis (the oral endoderm) take place. We argue that because the rates of photosynthesis and calcification per unit surface area are similar among the corals studied here, the areal iCA activity used to deliver Ci for these reactions should also be similar. The elevated iCA activity per unit volume of S. radians compared with that of the other species is probably due to the thinner effective tissue thickness in this species.

  12. Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus.

    PubMed

    Calhoun, Stacy; Zou, Enmin

    2016-03-01

    During the crustacean molting cycle, the exoskeleton is first mineralized in postmolt and intermolt and then presumably demineralized in premolt in order for epidermal retraction to occur. The mineralization process calls for divalent metal ions, such as Ca(2+) and Mg(2+) , and bicarbonate ions whereas protons are necessary for dissolution of carbonate salts. Carbonic anhydrase (CA) has been suggested to be involved in exoskeletal mineralization by providing bicarbonate ions through catalyzing the reaction of carbon dioxide hydration. However, results of earlier studies on the role of epidermal CA in metal incorporation in crustacean exoskeleton are not consistent. This study was aimed to provide further evidence to support the notion that epidermal CA is involved in exoskeletal mineralization using the blue crab, Callinectes sapidus (Rathbun 1896), as the model crustacean. Significant increases first in calcium and magnesium then in manganese post-ecdysis indicate significant metal deposition during postmolt and intermolt. Significant positive correlation between calcium or magnesium content and epidermal CA activity in postmolt and intermolt constitutes evidence that CA is involved in the mineralization of the crustacean exoskeleton. Additionally, we proposed a hypothetical model to describe the role of epidermal CA in both mineralization and demineralization of the exoskeleton based on the results of epidermal CA activity and exoskeletal metal content during the molting cycle. Furthermore, we found that the pattern of epidermal CA activity during the molting cycle of C. sapidus is similar to that of ecdysteroids reported for the same species, suggesting that epidermal CA activity may be under control of the molting hormones.

  13. Hypoxia-induced carbonic anhydrase IX as a target for cancer therapy: from biology to clinical use.

    PubMed

    Pastorek, Jaromir; Pastorekova, Silvia

    2015-04-01

    The tumor microenvironment includes a complicated network of physiological gradients contributing to plasticity of tumor cells and heterogeneity of tumor tissue. Hypoxia is a key component generating intratumoral oxygen gradients, which affect the cellular expression program and lead to therapy resistance and increased metastatic propensity of weakly oxygenated cell subpopulations. One of the adaptive responses of tumor cells to hypoxia involves the increased expression and functional activation of carbonic anhydrase IX (CA IX), a cancer-related cell surface enzyme catalyzing the reversible conversion of carbon dioxide to bicarbonate ion and proton. Via its catalytic activity, CA IX participates in regulation of intracellular and extracellular pH perturbations that result from hypoxia-induced changes in cellular metabolism producing excess of acid. Through the ability to regulate pH, CA IX also facilitates cell migration and invasion. In addition, CA IX has non-catalytic function in cell adhesion and spreading. Thus, CA IX endows tumor cells with survival advantages in hypoxia/acidosis and confers an increased ability to migrate, invade and metastasize. Accordingly, CA IX is expressed in a broad range of tumors, where it is associated with prognosis and therapy outcome. Its expression pattern and functional implications in tumor biology make CA IX a promising therapeutic target, which can be hit either by immunotherapy with monoclonal antibodies or with compounds inhibiting its enzyme activity. The first strategy has already reached the clinical trials, whereas the second one is still in preclinical testing. Both strategies indicate that CA IX can become a clinically useful anticancer target, but urge further efforts toward better selection of patients for immunotherapy and deeper understanding of tumor types, clinical situations and synthetic lethality interactions with other treatment approaches.

  14. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors

    PubMed Central

    Sedlakova, Olga; Svastova, Eliska; Takacova, Martina; Kopacek, Juraj; Pastorek, Jaromir; Pastorekova, Silvia

    2013-01-01

    Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons, and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX) is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades) and converting intracellular signals to extracellular effects on adhesion, proteolysis, and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia and/or acidosis

  15. Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO₂ sequestration.

    PubMed

    Oviya, M; Sukumaran, V; Giri, Sib Sankar

    2013-10-01

    The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K(m) and V(max) of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min⁻¹ mg⁻¹, respectively; whereas, the K(m) and V(max) of free enzyme were 18.26 mM and 434.78 μmol min⁻¹ mg⁻¹, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.

  16. Oxygen-18 exchange as a measure of accessibility of CO/sub 2/ and HCO/sub 3//sup -/ to carbonic anhydrase in Chlorella vulgaris (UTEX 263)

    SciTech Connect

    Tu, C.K.; Acevedo-Duncan, M.; Wynns, G.C.; Silverman, D.N.

    1986-04-01

    The exchange of /sup 18/O between CO/sub 2/ and H/sub 2/O in stirred suspensions of Chlorella vulgaris (UTEX 263) was measured using a membrane inlet to a mass spectrometer. The depletion of /sup 18/O from CO/sub 2/ in the fluid outside the cells provides a method to study CO/sub 2/ and HCO/sub 3//sup -/ kinetics in suspensions of algae that contain carbonic anhydrase since /sup 18/O loss to H/sub 2/O is catalyzed inside the cells but not in the external fluid. Low-CO/sub 2/ cells of Chlorella vulgaris (grown with air) were added to a solution containing /sup 18/O enriched CO/sub 2/ and HCO/sub 3//sup -/ with 2 to 15 millimolar total inorganic carbon. The observed depletion of /sup 18/O from CO/sub 2/ was biphasic and the resulting /sup 18/O content of CO/sub 2/ was much less than the /sup 18/O content of HCO/sub 3//sup -/ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO/sub 3//sup -/ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10/sup -4/ s/sup -1/ due to experimental errors. The Fick's law rate constant for entry of CO/sub 2/ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO/sub 2/ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO/sub 2/ entry into the cell or both. The CO/sub 2/ hydration activity inside the cells was 160 times the uncatalyzed CO/sub 2/ hydration rate.

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

    PubMed

    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.

  18. A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942.

    PubMed Central

    Fukuzawa, H; Suzuki, E; Komukai, Y; Miyachi, S

    1992-01-01

    To understand the CO2-concentrating mechanism in cyanobacteria, a genomic DNA fragment that complements a temperature-sensitive high-CO2 (5%)-requiring mutant of Synechococcus PCC7942 has been isolated. An open reading frame (ORF272) encoding a polypeptide of 272 amino acids (Mr, 30,184) was found within the genomic region located 20 kilobases downstream from the genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLS). Insertion of a kanamycin-resistance gene cartridge within the ORF272 in wild-type cells led to a high-CO2-requiring phenotype. Strains carrying a gene disabled by insertional mutagenesis accumulated inorganic carbon in the cells, but they could not fix it efficiently, even though ribulose-1,5-bisphosphate carboxylase activity was comparable to that of the wild-type strain. Therefore, the ORF272 was designated as a gene icfA, which is essential to inorganic carbon fixation. Furthermore, the predicted icfA gene product shared significant sequence similarities with plant chloroplast carbonic anhydrases (CAs) from pea (22%) and spinach (22%) and also with the Escherichia coli cynT gene product (31%), which was recently identified to be E. coli CA. These results indicate that the putative CA encoded by icfA is essential to photosynthetic carbon dioxide fixation in cyanobacteria and that plant chloroplast CAs may have evolved from a common ancestor of the prokaryotic CAs, which are distinct from mammalian CAs and Chlamydomonas periplasmic CAs. PMID:1584776

  19. Influence of amino acid replacement at position 198 on catalytic properties of zinc-bound water in human carbonic anhydrase III.

    PubMed

    LoGrasso, P V; Tu, C; Chen, X; Taoka, S; Laipis, P J; Silverman, D N

    1993-06-01

    Carbonic anhydrase III, found predominantly in skeletal muscle, is the least efficient of the mammalian carbonic anhydrases in catalyzing the hydration of CO2. Phenylalanine-198 is located on the hydrophobic side of the active-site cavity with its phenyl ring in the proximity of the catalytically active zinc-bound water. We replaced phenylalanine-198 in human carbonic anhydrase III with seven other amino acids (Ala, Asn, Asp, His, Leu, Tyr, Val) using site-directed mutagenesis. The catalytic properties of these enzymes were determined by stopped-flow spectrophotometry, and the exchange of 18O between CO2 and water was measured by mass spectrometry. All of the mutants had maximal values of kcat/Km for the hydration of CO2 enhanced, and five of the mutants had the pKa of the zinc-bound water increased compared with the wild-type enzyme. The largest effects were observed with the replacement Phe-198-->Asp which increased the maximal kcat/Km 140-fold and increased the pKa of the zinc-bound water from near 5 to 9.2. A Brønsted correlation was observed between log(kcat/Km) for hydration of CO2 and the pKa of the zinc-bound water (correlation coefficient r = 0.92); in addition, this pKa was inversely correlated with hydrophobicity of the residue at 198 (correlation coefficient r = -0.83). A direct correlation between the logarithm of the maximal kcat/Km for hydration and the logarithm of the pH-independent value of Ki for inhibition by cyanate (r = 0.95) indicated that the effect of the mutations at residue 198 occurred in large part by enhancement of the rate of dissociation of the enzyme-bicarbonate complex. PMID:8504098

  20. The γ-carbonic anhydrase subcomplex of mitochondrial complex I is essential for development and important for photomorphogenesis of Arabidopsis.

    PubMed

    Wang, Qin; Fristedt, Rikard; Yu, Xuhong; Chen, Zugen; Liu, Hongtao; Lee, Yurhee; Guo, Hongwei; Merchant, Sabeeha S; Lin, Chentao

    2012-11-01

    Complex I (NADH:ubiquinone oxidoreductase) is the entry point for electrons into the respiratory electron transport chain; therefore, it plays a central role in cellular energy metabolism. Complex I from different organisms has a similar basic structure. However, an extra structural module, referred to as the γ-carbonic anhydrase (γCA) subcomplex, is found in the mitochondrial complex I of photoautotrophic eukaryotes, such as green alga and plants, but not in that of the heterotrophic eukaryotes, such as fungi and mammals. It has been proposed that the γCA subcomplex is required for the light-dependent life style of photoautotrophic eukaryotes, but this hypothesis has not been successfully tested. We report here a genetic study of the genes γCAL1 and γCAL2 that encode two subunits of the γCA subcomplex of mitochondrial complex I. We found that mutations of γCAL1 and γCAL2 in Arabidopsis (Arabidopsis thaliana) result in defective embryogenesis and nongerminating seeds, demonstrating the functional significance of the γCA subcomplex of mitochondrial complex I in plant development. Surprisingly, we also found that reduced expression of γCAL1 and γCAL2 genes altered photomorphogenic development. The γcal1 mutant plant expressing the RNA interference construct of the γCAL2 gene showed a partial constitutive photomorphogenic phenotype in young seedlings and a reduced photoperiodic sensitivity in adult plants. The involvement of the γCA subcomplex of mitochondrial complex I in plant photomorphogenesis and the possible evolutionary significance of this plant-specific mitochondrial protein complex are discussed.

  1. The effect of carbonic anhydrase inhibitors on secretion by the parotid and mandibular glands of red kangaroos Macropus rufus.

    PubMed

    Beal, A M

    1991-01-01

    The effects of carbonic anhydrase inhibitors on secretion by macropodine parotid and mandibular glands were investigated using anaesthetized red kangaroos. In the parotid gland, acetazolamide (500 mumol.l-1) reduced a stable acetylcholine-evoked, half-maximal flow rate of 2.02 +/- 0.034 to 0.27 +/- 0.023 ml.min-1 (87% reduction). Concurrently, salivary bicarbonate concentration and secretion fell (129.4 +/- 1.46 to 80.9 +/- 1.63 mmol.l-1 and 264.8 +/- 7.96 to 22.3 +/- 2.30 mumol.min-1, respectively), phosphate and chloride concentrations rose (14.0 +/- 0.79 to 27.6 +/- 0.85 mmol.l-1 and 5.6 +/- 0.25 to 27.5 +/- 1.32 mmol.l-1, respectively), sodium concentration and osmolality were unaltered, and potassium concentration fell (8.8 +/- 0.33 to 6.4 +/- 0.29 mmol.l-1). High-rate cholinergic stimulation during acetazolamide blockade was unable to increase salivary flow beyond 11 +/- 0.9% of that for equivalent unblocked control stimulation. However, superimposition of isoprenaline infusion on the acetylcholine stimulation caused a three-fold increase in the blocked flow rate. These treatments were accompanied by small increases in salivary phosphate and chloride concentrations but not bicarbonate concentration. Methazolamide infusion caused similar changes in parotid secretion. In the mandibular gland, acetazolamide infusion had no effect on salivary flow rate during either low- or high-level acetylcholine stimulation. Acetazolamide caused no alterations in salivary electrolyte secretion at low flow rates, but curtailed the rise in bicarbonate concentration associated with high-level acetylcholine stimulation. Acetazolamide administration did not affect the increase in salivary flow rate associated with isoprenaline infusion, but did block the concomitant increase in bicarbonate concentration and secretion substantially.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII.

    PubMed

    Cvijetić, Ilija N; Tanç, Muhammet; Juranić, Ivan O; Verbić, Tatjana Ž; Supuran, Claudiu T; Drakulić, Branko J

    2015-08-01

    Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX and XII was evaluated by a stopped-flow CO2 hydrase assay. These compounds exerted a clear, selective inhibition of hCA IX and XII over hCAI and II, with Ki in two to one digit micromolar concentrations (4-50 μM). Derivatives bearing bulkier substituents in para-position of the phenyl ring inhibited hCA XII at one-digit micromolar concentrations, while derivatives having alkyl substituents in both ortho- and meta-positions inhibited hCA IX with Kis ranging between 5 and 25 μM. Results of docking experiments offered a rational explanation on the selectivity of these compounds toward CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII inhibitors. By examining the active sites of these four isoforms with GRID generated molecular-interaction fields, striking differences between hCA XII and the other three isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly different in CA XII active site, comparing to other three isoforms studied. To the best of our knowledge such an observation was not reported in literature so far. Considering the selectivity of these carboxylates towards membrane-associated over cytosolic CA isoforms, the title compounds could be useful for the development of isoform-specific non-sulfonamide CA inhibitors.

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

    PubMed

    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). There was no correlation among the SNPs' frequencies and OHI-S, PI, GI, SFR, and SpH (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

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

    PubMed

    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). There was no correlation among the SNPs' frequencies and OHI-S, PI, GI, SFR, and SpH (p > 0.05). CA VI SNP (rs2274327) had no statistically significant association with OHI-S, PI, GI, SFR, and SpH in the children.

  5. High Stromal Carbonic Anhydrase IX Expression Is Associated With Decreased Survival in p16-Negative Head-and-Neck Tumors

    SciTech Connect

    Brockton, Nigel; Dort, Joseph; Lau, Harold; Hao, Desiree; Brar, Sony; Klimowicz, Alexander; Petrillo, Stephanie; Diaz, Roman; Doll, Corinne; Magliocco, Anthony

    2011-05-01

    Purpose: Head-and-neck squamous cell carcinoma (HNSCC) is the fifth most common malignancy worldwide. Alcohol use and tobacco use are the most established risk factors; however, human papilloma virus (HPV) infection is a major risk factor for a subset of HNSCCs. Although HPV-positive tumors typically present at a more advanced stage at diagnosis, they are associated with a better prognosis. Tumor hypoxia confers poor prognosis and treatment failure, but direct tumor oxygen measurement is challenging. Endogenous markers of hypoxia (EMHs) have been proposed but have not replicated the prognostic utility of direct oxygen measurement. The expression of endogenous markers of hypoxia may be influenced by oxygen-independent factors, such as the HPV status of the tumor. Methods and Materials: Consecutive cases of locally advanced HNSCC, treated with a uniform regimen of combined radiotherapy and chemotherapy, were identified. Tissue microarrays were assembled from triplicate 0.6-mm cores of archived tumor tissue. HPV status was inferred from semiquantitative p16 immunostaining and directly measured by use of HPV-specific chromogenic in situ hybridization and polymerase chain reaction. Automated quantitative fluorescent immunohistochemistry was conducted to measure epithelial and stromal expression of carbonic anhydrase IX (CAIX) and glucose transporter 1 (GLUT1). Results: High stromal CAIX expression was associated with significantly reduced overall survival (p = 0.03) in patients with p16-negative tumors. Conclusions: This is the first study to use quantitative immunohistochemistry to examine endogenous markers of hypoxia stratified by tumor p16/HPV status. Assessment of CAIX expression in p16-negative HNSCC could identify patients with the least favorable prognosis and inform therapeutic strategies.

  6. Chloride dysregulation and inhibitory receptor blockade yield equivalent disinhibition of spinal neurons yet are differentially reversed by carbonic anhydrase blockade.

    PubMed

    Lee, Kwan Yeop; Prescott, Steven A

    2015-12-01

    Synaptic inhibition plays a key role in processing somatosensory information. Blocking inhibition at the spinal level is sufficient to produce mechanical allodynia, and many neuropathic pain conditions are associated with reduced inhibition. Disinhibition of spinal neurons can arise through decreased GABAA/glycine receptor activation or through dysregulation of intracellular chloride. We hypothesized that these distinct disinhibitory mechanisms, despite all causing allodynia, are differentially susceptible to therapeutic intervention. Specifically, we predicted that reducing bicarbonate efflux by blocking carbonic anhydrase with acetazolamide (ACTZ) would counteract disinhibition caused by chloride dysregulation without affecting normal inhibition or disinhibition caused by GABAA/glycine receptor blockade. To test this, responses to innocuous tactile stimulation were recorded in vivo from rat superficial dorsal horn neurons before and after different forms of pharmacological disinhibition and again after application of ACTZ. Blocking GABAA or glycine receptors caused hyperresponsiveness equivalent to that caused by blocking the potassium chloride cotransporter KCC2, but, consistent with our predictions, only disinhibition caused by KCC2 blockade was counteracted by ACTZ. ACTZ did not alter responses of neurons with intact inhibition. As pathological downregulation of KCC2 is triggered by brain-derived neurotrophic factor, we also confirmed that ACTZ was effective against brain-derived neurotrophic factor-induced hyperresponsiveness. Our results argue that intrathecal ACTZ has antiallodynic effects only if allodynia arises through chloride dysregulation; therefore, behavioral evidence that ACTZ is antiallodynic in nerve-injured animals affirms the contribution of chloride dysregulation as a key pathological mechanism. Although different disinhibitory mechanisms are not mutually exclusive, these results demonstrate that their relative contribution dictates which

  7. Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.

    PubMed

    Krishnan, Devishree; Liu, Lei; Wiebe, Shane A; Casey, Joseph R; Cordat, Emmanuelle; Alexander, R Todd

    2015-08-15

    Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.

  8. Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity

    PubMed Central

    Bootorabi, Fatemeh; Jänis, Janne; Valjakka, Jarkko; Isoniemi, Sari; Vainiotalo, Pirjo; Vullo, Daniela; Supuran, Claudiu T; Waheed, Abdul; Sly, William S; Niemelä, Onni; Parkkila, Seppo

    2008-01-01

    Background Acetaldehyde, the first metabolite of ethanol, can generate covalent modifications of proteins and cellular constituents. However, functional consequences of such modification remain poorly defined. In the present study, we examined acetaldehyde reaction with human carbonic anhydrase (CA) isozyme II, which has several features that make it a suitable target protein: It is widely expressed, its enzymatic activity can be monitored, its structural and catalytic properties are known, and it contains 24 lysine residues, which are accessible sites for aldehyde reaction. Results Acetaldehyde treatment in the absence and presence of a reducing agent (NaBH3(CN)) caused shifts in the pI values of CA II. SDS-PAGE indicated a shift toward a slightly higher molecular mass. High-resolution mass spectra of CA II, measured with and without NaBH3(CN), indicated the presence of an unmodified protein, as expected. Mass spectra of CA II treated with acetaldehyde revealed a modified protein form (+26 Da), consistent with a "Schiff base" formation between acetaldehyde and one of the primary NH2 groups (e.g., in lysine side chain) in the protein structure. This reaction was highly specific, given the relative abundance of over 90% of the modified protein. In reducing conditions, each CA II molecule had reacted with 9–19 (14 on average) acetaldehyde molecules (+28 Da), consistent with further reduction of the "Schiff bases" to substituted amines (N-ethyllysine residues). The acetaldehyde-modified protein showed decreased CA enzymatic activity. Conclusion The acetaldehyde-derived modifications in CA II molecule may have physiological consequences in alcoholic patients. PMID:19036170

  9. Histopathological Determinants of Tumor Resistance: A Special Look to the Immunohistochemical Expression of Carbonic Anhydrase IX in Human Cancers

    PubMed Central

    Ilardi, G.; Zambrano, N.; Merolla, F.; Siano, M.; Varricchio, S.; Vecchione, M.; De Rosa, G.; Mascolo, M.; Staibano, S.

    2014-01-01

    Intrinsic and acquired drug resistance of tumor cells still causes the failure of treatment regimens in advanced human cancers. It may be driven by intrinsic tumor cells features, or may also arise from micro environmental influences. Hypoxia is a microenvironment feature associated with the aggressiveness and metastasizing ability of human solid cancers. Hypoxic cancer cells overexpress Carbonic Anhydrase IX (CA IX). CA IX ensures a favorable tumor intracellular pH, while contributing to stromal acidosis, which facilitates tumor invasion and metastasis. The overexpression of CA IX is considered an epiphenomenon of the presence of hypoxic, aggressive tumor cells. Recently, a relationship between CA IX overexpression and the cancer stem cells (CSCs) population has been hypothesized. CSCs are strictly regulated by tumor hypoxia and drive a major non-mutational mechanism of cancer drug-resistance. We reviewed the current data concerning the role of CA IX overexpression in human malignancies, extending such information to the expression of the stem cells markers CD44 and nestin in solid cancers, to explore their relationship with the biological behavior of tumors. CA IX is heavily expressed in advanced tumors. A positive trend of correlation between CA IX overexpression, tumor stage/grade and poor outcome emerged. Moreover, stromal CA IX expression was associated with adverse events occurrence, maybe signaling the direct action of CA IX in directing the mesenchymal changes that favor tumor invasion; in addition, membranous/cytoplasmic co-overexpression of CA IX and stem cells markers were found in several aggressive tumors. This suggests that CA IX targeting could indirectly deplete CSCs and counteract resistance of solid cancers in the clinical setting. PMID:23992304

  10. Carbonic anhydrase III and four-and-a-half LIM protein 1 are preferentially oxidized with muscle unloading

    PubMed Central

    Chen, Chiao-nan; Ferrington, Deborah A.; Thompson, LaDora V.

    2008-01-01

    The identities of proteins that show disuse-related changes in the content of oxidative modification are unknown. Furthermore, it is unknown whether the global accumulation of oxidized proteins is greater in aged animals with muscle disuse. The purposes of this study are 1) to identify the exact proteins that show disuse-related changes in oxidation levels and 2) to test the hypothesis that the global accumulation of oxidized proteins with muscle disuse would be greater in aged animals. Adult and old rats were randomized into four groups: weight bearing and 3, 7, or 14 days of hindlimb unloading. Soleus muscles were harvested to investigate the protein oxidation with unloading. Slot blot, SDS-PAGE, and Western blot analyses were used to detect the accumulation of 4-hydroxy-2-nonenol (HNE)- and nitrotyrosine (NT)-modified proteins. Matrix-assisted laser desorption ionization-time of flight and tandem mass spectroscopy were used to identify modified proteins. We found that global HNE- and NT-modified proteins accumulated significantly with aging but not with muscle unloading. Two HNE and NT target proteins, four-and-a-half LIM protein 1 (FHL1) and carbonic anhydrase III (CAIII), showed changes in the oxidation levels with muscle unloading. The changes in the oxidation levels happened to adult rats but not old rats. However, old rats had higher baseline levels of HNE-modified FHL1. In summary, the data suggest that the muscle unloading-related changes of protein oxidation are more significant in specific proteins and that the changes are age related. PMID:18756007

  11. Estrogen and progesterone differentially regulate carbonic anhydrase II, III, IX, XII, and XIII in ovariectomized rat uteri.

    PubMed

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-01-01

    Changes in the uterus expression of carbonic anhydrase (CA) II, III, IX, XII, and XIII were investigated under the influence of sex-steroids in order to elucidate mechanisms underlying differential effects of these hormones on uterine pH. Uteri of ovariectomised rats receiving over three days either vehicle, estrogen, or progesterone or three days estrogen followed by three days either vehicle or progesterone were harvested. Messenger RNA (mRNA) and protein levels were quantified by real-time PCR and Western blotting, respectively. The distribution of CA isoenzymes proteins were examined by immunohistochemistry. The levels of CAII, III, XII, and XIII mRNAs and proteins were elevated while levels of CAIX mRNA and protein were reduced following progesterone-only and estrogen plus progesterone treatment, compared to the control and estrogen plus vehicle, respectively. Following estrogen treatment, expression of CAII, IX, XII, and CAXIII mRNAs and proteins were reduced, but remained at a level higher than control, except for CAIX, where its level was higher than the control and following progesterone treatment. Under progesterone-only and estrogen plus progesterone influences, high levels of CAII, III, XII, and XIII were observed in uterine lumenal and glandular epithelia and myometrium. However, a high level of CAIX was observed only under the influence of estrogen at the similar locations. In conclusion, high expression of CAII, III, XII, and XIII under the influence of progesterone and estrogen plus progesterone could result in the reduction of uterine tissue and fluid pH; however, the significance of high levels of CAIX expression under the influence of estrogen remains unclear. PMID:26709452

  12. Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

    PubMed Central

    Lotlikar, Shalaka R.; Hnatusko, Shane; Dickenson, Nicholas E.; Choudhari, Shyamal P.; Picking, Wendy L.

    2013-01-01

    Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28–45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn2+ and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19–26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism. PMID:23728627

  13. Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation▿

    PubMed Central

    Kalloniati, Chrysanthi; Tsikou, Daniela; Lampiri, Vasiliki; Fotelli, Mariangela N.; Rennenberg, Heinz; Chatzipavlidis, Iordanis; Fasseas, Costas; Katinakis, Panagiotis; Flemetakis, Emmanouil

    2009-01-01

    Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3− produced by CAA1. PMID:19218391

  14. Carbonic anhydrase activators: the first activation study of the human secretory isoform VI with amino acids and amines.

    PubMed

    Nishimori, Isao; Onishi, Saburo; Vullo, Daniela; Innocenti, Alessio; Scozzafava, Andrea; Supuran, Claudiu T

    2007-08-01

    The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4 x 10(5)s(-1) and k(cat)/K(M) of 4.9 x 10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of amino acids and amines were shown to act as CA VI activators, with variable efficacies. l-His, l-Trp, and dopamine showed weak CA VI activating effects (K(A)s in the range of 21-42 microM), whereas d-His, d-Phe, l-DOPA, l-Trp, serotonin, and some pyridyl-alkylamines were better activators, with K(A)s in the range of 13-19 microM. The best CA VI activators were l-Phe, d-DOPA, l-Tyr, 4-amino-l-Phe, and histamine, with K(A)s in the range of 1.23-9.31 microM. All these activators enhance k(cat), having no effect on K(M), participating thus in the rate determining step in the catalytic cycle, the proton transfer reactions between the enzyme active site and the environment. PMID:17499996

  15. Thermodynamics of binding of a sulfonamide inhibitor to metal-mutated carbonic anhydrase as studied by affinity capillary electrophoresis.

    PubMed

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko

    2015-09-01

    By affinity capillary electrophoresis (ACE), the thermodynamic binding constants of a sulfonamide (SA) inhibitor to bovine carbonic anhydrase II (CA) and metal mutated variants (M-CAs) were evaluated. 1-(4-Aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate was used as the SA in the electrophoretic buffer for ACE. The Scatchard analysis of the dependence of the electrophoretic mobility of native CA on the SA concentration provided the binding constant to be Kb=(2.29±0.05)×10(6) M(-1) (at pH8.4, 25°C). On the other hand, apoCA showed far smaller value [Kb=(3.76±0.14)×10(2) M(-1)], suggesting that the coordination of SA to the Zn(II) center controlled the binding thermodynamics. The ACE of M-CAs showed the same behaviors as native CA but with different Kb values. For example, Co-CA adopting the same tetrahedral coordination geometry as native CA exhibited the largest Kb value [(2.55±0.05)×10(6) M(-1)] among the M-CAs. In contrast, Mn- and Ni-CA, which adopted the octahedral coordination geometry, had Kb values that were about two orders of magnitude lower. Because the hydrophobic cavity of CA around the active center pre-organized the orientation of SA, thereby fixing the ligating NH(-) moiety to the apex of the tetrahedron supported by three basal His3 of CA, metals such as Zn and Co at the center of M-CA gave the most stable CA-SA complex. However, pre-organization was not favored for octahedral geometry. Thus, pre-organization of SA was the key to facilitating the tetrahedral coordination geometry of the Zn(II) active center of CA.

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

    PubMed

    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

  17. Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6-dihydropyrimidine-benzenesulfonamides acting as carbonic anhydrase inhibitors.

    PubMed

    Vullo, Daniela; Supuran, Claudiu T; Scozzafava, Andrea; De Simone, Giuseppina; Monti, Simona Maria; Alterio, Vincenzo; Carta, Fabrizio

    2016-08-15

    Herein we report an in vitro kinetic evaluation against the most relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms (I, II, IX and XII) of a small series of lactate dehydrogenase (LDH, EC 1.1.1.27) inhibitors. All compounds contain a primary sulfonamide zinc-binding group (ZBG) substituted with the 2-thio-6-oxo-1,6-dihydropyrimidine scaffold. By means of X-ray crystallographic experiments we explored the ligand-enzyme binding modes, thus highlighting the contribution of the 2-thio-6-oxo-1,6-dihydropyrimidine moiety to the stabilization of the complex. PMID:27316543

  18. Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole, a structural and analog of acetazolamide, show interesting carbonic anhydrase inhibitory properties, diuretic, and anticonvulsant action.

    PubMed

    Diaz, Jorge R A; Camí, Gerardo Enrique; Liu-González, Malva; Vega, Daniel R; Vullo, Daniela; Juárez, Américo; Pedregosa, José C; Supuran, Claudiu T

    2016-12-01

    Three salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole (Hats) were prepared and characterized by physico-chemical methods. The p-toluensulfonate, the methylsulfonate, and the chlorhydrate monohydrate salts of Hats were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) and as anticonvulsants and diuretics, since many CAIs are clinically used as pharmacological agents. The three Hats salts exhibited diuretic and anticonvulsant activities with little neurotoxicity. The human (h) isoforms hCA I, II, IV, VII, IX, and XII were inhibited in their micromolar range by these salts, whereas pathogenic beta and gamma CAs showed similar, weak inhibitory profiles.

  19. 1,2-Benzisothiazole Derivatives Bearing 4-, 5-, or 6-Alkyl/arylcarboxamide Moieties Inhibit Carbonic Anhydrase Isoform IX (CAIX) and Cell Proliferation under Hypoxic Conditions.

    PubMed

    Coviello, Vito; Marchi, Beatrice; Sartini, Stefania; Quattrini, Luca; Marini, Anna Maria; Simorini, Francesca; Taliani, Sabrina; Salerno, Silvia; Orlandi, Paola; Fioravanti, Anna; Desidero, Teresa Di; Vullo, Daniela; Da Settimo, Federico; Supuran, Claudiu T; Bocci, Guido; La Motta, Concettina

    2016-07-14

    Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated. PMID:27305384

  20. Metal Complexes of 1,3,4-Thiadiazole-2,5-Disulfonamide are Strong Dual Carbonic Anhydrase Inhibitors, although the Ligand Possesses very Weak such Properties

    PubMed Central

    Supuran, Claudiu T.

    1995-01-01

    Coordination compounds of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with 1,3,4-thiadiazole-2,5-disulfonamide as ligand were synthesized and characterized by IR and UV spectroscopy, conductimetry and thermogravimetry. The parent ligand is a very weak carbonic anhydrase (CA) inhibitor, although it constituted the lead for developing important classes of diuretics. The complex derivatives behave as much stronger CA inhibitors, with IC50 values around 10−8M against isozyme CA II, and 10−7 M against isozyme CAI. PMID:18472784

  1. Metal Complexes of 1,3,4-Thiadiazole-2,5-Disulfonamide are Strong Dual Carbonic Anhydrase Inhibitors, although the Ligand Possesses very Weak such Properties.

    PubMed

    Supuran, C T

    1995-01-01

    Coordination compounds of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with 1,3,4-thiadiazole-2,5-disulfonamide as ligand were synthesized and characterized by IR and UV spectroscopy, conductimetry and thermogravimetry. The parent ligand is a very weak carbonic anhydrase (CA) inhibitor, although it constituted the lead for developing important classes of diuretics. The complex derivatives behave as much stronger CA inhibitors, with IC(50) values around 10(-8)M against isozyme CA II, and 10(-7) M against isozyme CAI.

  2. Single-eye trial of a topical carbonic anhydrase inhibitor versus intravitreal bevacizumab for the treatment of taxane drug-induced cystoid macula oedema.

    PubMed

    Hassall, Mark M; Andrew, Nicholas Howard

    2016-04-19

    Taxanes are a class of microtubule stabilising agents used to treat a wide range of malignancies. Taxane drug-induced cystoid macula oedema (TDICMO) is a known but rare complication of therapy. First reported with Docetaxel in 2003 and Paclitaxel in 2007, there are currently less than 20 cases of TDICMO in the literature. Although most cases resolve following taxane cessation, several authors have tried using carbonic anhydrase inhibitors or intravitreal bevacizumab to accelerate resolution or when taxane therapy cannot be discontinued. We report the first published case of TDICMO treated with a single-eye trial of topical dorzolamide versus intravitreal bevacizumab.

  3. Neutron structure of human carbonic anhydrase II: A hydrogen bonded water network switch is observed between pH 7.8 and 10.0.

    SciTech Connect

    Fisher, Zoe; Langan, Paul; Mustyakimov, Marat; Kovalevsky, Andrey

    2011-01-01

    The neutron structure of wild type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 resolution. Detailed analysis and comparison to the previous determined structure at pH 10.0 shows important differences in protonation of key catalytic residues in the active site as well as a rearrangement of the hydrogen bonded water network. For the first time, a completed hydrogen bonded network stretching from the Zn-bound solvent to the proton shuttling residue His64 has been directed observed.

  4. Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    PubMed

    Ferraroni, Marta; Del Prete, Sonia; Vullo, Daniela; Capasso, Clemente; Supuran, Claudiu T

    2015-12-01

    Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA from Vibrio cholerae (VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 × 10(5) s(-1) and a kcat/Km of 4.1 × 10(7) M(-1) s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Ki of 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.

  5. Comparison of sulfamate and sulfamide groups for the inhibition of carbonic anhydrase-II by using topiramate as a structural platform.

    PubMed

    Maryanoff, Bruce E; McComsey, David F; Costanzo, Michael J; Hochman, Coralie; Smith-Swintosky, Virginia; Shank, Richard P

    2005-03-24

    This paper examines the relative effectiveness of sulfamate and sulfamide groups for the inhibition of carbonic anhydrase-II (CA-II). Topiramate (1) and its sulfamide analogue 4, and 4,5-cyclic sulfate 6 and its sulfamide analogue 5, were compared for inhibition of human CA-II. A colorimetric assay, based on the pH shift that accompanies hydration of carbon dioxide, and an esterase assay were used. For these bioisosteric pairs, 1/4 and 6/5, the sulfamate compound was markedly more potent than its sulfamide counterpart. A similar, large difference in potency was also observed for the sulfamate/sulfamide pairs 14/15 and 16/17. These results indicate that the sulfamide moiety is not particularly suitable for obtaining potent carbonic anhydrase inhibition. A discussion of this structure-activity relationship with respect to the interactions of 1 and 6 with CA-II from published X-ray data is presented. A metabolic acidosis study was performed in rats with 1, 4, 6, and 2, and the results are discussed with respect to the degree of inhibition of CA-II in vivo. PMID:15771438

  6. Biomimetic CO₂ sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials.

    PubMed

    Sharma, Anjana; Bhattacharya, Abhishek; Shrivastava, Ankita

    2011-04-01

    The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan-KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan-NH(4)OH beads. A maximum increase of 1.08-1.18 fold stability between 35 and 55°C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan-KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan-KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate. PMID:22112959

  7. Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774).

    PubMed

    Pederzoli, Aurora; Mandrioli, Mauro; Mola, Lucrezia

    2014-04-01

    Sequencing of the amphioxus genome revealed that it contains a basic set of chordate genes involved in development and cell signaling. Despite the availability of genomic data, up till now no studies have been addressed on the comprehension of the amphioxus osmoregulation. Using primers designed on Branchiostoma floridae carbonic anhydrase (CA) II, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase, a 100bp long region, containing the protein region recognized by the respective antibodies, has been amplified and sequenced in B. lanceolatum indicating the presence of hortologous V-ATPase, CFTR and carbonic anhydrase II genes in Branchiostoma lanceolatum. Immunohistochemical results showed that all three transporting proteins are expressed in almost 90% of epithelial cells of the skin in B. lanceolatum adults with a different degree of positivity in different regions of body wall and with a different localization in the cells. The comparison of results between young and adult lancelets showed that the distribution of these transporters is quite different. Indeed, in the young specimens the expression pattern of all tested molecules appears concentrated at the gut level, whereas in adult the gut loses its key role that is mostly supported by skin.

  8. Synthesis, characterization, antimicrobial activity and carbonic anhydrase enzyme inhibitor effects of salicilaldehyde-N-methyl p-toluenesulfonylhydrazone and its Palladium(II), Cobalt(II) complexes

    NASA Astrophysics Data System (ADS)

    Alyar, Saliha; Adem, Şevki

    2014-10-01

    We report the synthesis of the ligand, salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Pd(II) and Co(II) metal complexes were synthesized for the first time. The structure of the ligand and their complexes were investigated using elemental analysis, magnetic susceptibility, molar conductance and spectral (IR, NMR and LC-MS) measurements. Salptsmh has also been characterized by single crystal X-ray diffraction. 1H and 13C shielding tensors for crystal structure were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl3. The complexes were found to have general composition [ML2]. The results of elemental analysis showed 1:2 (metal/ligand) stoichiometry for all the complex. Magnetic and spectral data indicate a square planar geometry for Pd(II) complex and a distorted tetrahedral geometry for Co(II) complexes. The ligand and its metal chelates have been screened for their antimicrobial activities using the disk diffusion method against the selected Gram positive bacteria: Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Gram negative bacteria: Eschericha coli, Pseudomonas aeruginosa, Klebsiella pneumonia. The inhibition activities of these compounds on carbonic anhydrase II (CA II) and carbonic anhydrase I (CA I) have been investigated by comparing IC50 and Ki values and it has been found that Pd(II) complex have more enzyme inhibition efficiency than salptsmh and Co(II) complex.

  9. Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

    PubMed

    Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

    2009-10-01

    Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

  10. Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774).

    PubMed

    Pederzoli, Aurora; Mandrioli, Mauro; Mola, Lucrezia

    2014-04-01

    Sequencing of the amphioxus genome revealed that it contains a basic set of chordate genes involved in development and cell signaling. Despite the availability of genomic data, up till now no studies have been addressed on the comprehension of the amphioxus osmoregulation. Using primers designed on Branchiostoma floridae carbonic anhydrase (CA) II, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase, a 100bp long region, containing the protein region recognized by the respective antibodies, has been amplified and sequenced in B. lanceolatum indicating the presence of hortologous V-ATPase, CFTR and carbonic anhydrase II genes in Branchiostoma lanceolatum. Immunohistochemical results showed that all three transporting proteins are expressed in almost 90% of epithelial cells of the skin in B. lanceolatum adults with a different degree of positivity in different regions of body wall and with a different localization in the cells. The comparison of results between young and adult lancelets showed that the distribution of these transporters is quite different. Indeed, in the young specimens the expression pattern of all tested molecules appears concentrated at the gut level, whereas in adult the gut loses its key role that is mostly supported by skin. PMID:24220283

  11. Effect of lecithin and MgCO3 as additives on the enzymatic activity of carbonic anhydrase encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres.

    PubMed

    Sandor, Maryellen; Riechel, Alex; Kaplan, Ian; Mathiowitz, Edith

    2002-02-15

    A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme. PMID:11960690

  12. Mass spectrometric determination of HCO3- permeability and carbonic anhydrase activity in intact guinea-pig colon epithelium.

    PubMed

    Böllert, P; Peters, T; von Engelhardt, W; Gros, G

    1997-08-01

    1. A mass spectrometric method originally used in red blood cells was applied to suspensions of isolated colonocytes and intact colonic epithelium to measure the exchange of 18O between HCO3-, CO2 and H2O to determine intracellular carbonic anhydrase activity (Ai) and membrane bicarbonate permeability (P). 2. In suspensions of isolated guinea-pig colon epithelial cells, colonocytes, we found significantly higher values of Ai and P for cells derived from the proximal colon than for cells from the distal colon. In the case of Ai, this confirms earlier reports. 3. When the 18O exchange process was observed across the mucosal (apical) side of intact colon mucosa, the estimated values of Ai were identical to those obtained for isolated colonocytes, for both the proximal and the distal part of the colon. This is considered to be strong evidence that this method can be applied to a layer of intact epithelium as well as to cell suspensions. 4. The values of P obtained from the apical side of intact colon mucosa were 6 times higher than those estimated from measurements with isolated colonocytes. This indicates that the basolateral membrane of colon epithelium, which participates in the 18O exchange process in isolated colonocytes but not in the 18O exchange process across the apical side of intact mucosa, has a markedly lower bicarbonate permeability than the apical membrane. 5. When the 18O exchange process was observed across the serosal (basolateral) side of intact colon mucosa, the P values, as expected, were low compared with the apical side of intact mucosa. However, rather unexpectedly, the Ai values derived from these measurements were 2-3 times lower than those obtained with isolated colonocytes. It appears possible that the latter finding is an artifact due to the submucosal tissue markedly slowing down CO2 diffusion from the bathing medium into the epithelial cells, thus causing an apparent fall in Ai. 6. Ai decreased and P increased with increasing temperature

  13. Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells

    SciTech Connect

    Vordermark, Dirk . E-mail: vordermark_d@klinik.uni-wuerzburg.de; Kaffer, Anja; Riedl, Susanne; Katzer, Astrid; Flentje, Michael

    2005-03-15

    Purpose: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. Methods and materials: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. Results: Hypoxia-induced CA IX protein expression was seen after >6 h at {<=}5% O{sub 2}, and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. Conclusion: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1{alpha}. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro

  14. Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

    PubMed

    Zhou, Wei; Sui, Zhenghong; Wang, Jinguo; Hu, Yiyi; Kang, Kyoung Ho; Hong, Hye Ran; Niaz, Zeeshan; Wei, Huihui; Du, Qingwei; Peng, Chong; Mi, Ping; Que, Zhou

    2016-06-01

    There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae. PMID:26960545

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

    PubMed

    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

  16. Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

    PubMed

    Zhou, Wei; Sui, Zhenghong; Wang, Jinguo; Hu, Yiyi; Kang, Kyoung Ho; Hong, Hye Ran; Niaz, Zeeshan; Wei, Huihui; Du, Qingwei; Peng, Chong; Mi, Ping; Que, Zhou

    2016-06-01

    There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

  17. Carborane-Based Carbonic Anhydrase Inhibitors: Insight into CAII/CAIX Specificity from a High-Resolution Crystal Structure, Modeling, and Quantum Chemical Calculations

    PubMed Central

    Mader, Pavel; Pecina, Adam; Cígler, Petr; Lepšík, Martin; Šícha, Václav; Hobza, Pavel; Grüner, Bohumír; Fanfrlík, Jindřich; Brynda, Jiří; Řezáčová, Pavlína

    2014-01-01

    Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively. PMID:25309911

  18. Metalloprotein-inhibitor binding: human carbonic anhydrase II as a model for probing metal-ligand interactions in a metalloprotein active site.

    PubMed

    Martin, David P; Hann, Zachary S; Cohen, Seth M

    2013-11-01

    An ever-increasing number of metalloproteins are being discovered that play essential roles in physiological processes. Inhibitors of these proteins have significant potential for the treatment of human disease, but clinical success of these compounds has been limited. Herein, zinc(II)-dependent metalloprotein inhibitors in clinical use are reviewed, and the potential for using novel metal-binding groups (MBGs) in the design of these inhibitors is discussed. By using human carbonic anhydrase II as a model system, the nuances of MBG-metal interactions in the context of a protein environment can be probed. Understanding how metal coordination influences inhibitor binding may help in the design of new therapeutics targeting metalloproteins.

  19. Carbonic anhydrase inhibitors: benzenesulfonamides incorporating cyanoacrylamide moieties are low nanomolar/subnanomolar inhibitors of the tumor-associated isoforms IX and XII.

    PubMed

    Alafeefy, Ahmed M; Isik, Semra; Abdel-Aziz, Hatem A; Ashour, Abdelkader E; Vullo, Daniela; Al-Jaber, Nabila A; Supuran, Claudiu T

    2013-03-15

    A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues) have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new benzenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less effective as inhibitors of CA I and II. The structure-activity relationship for this class of effective CA inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very potent CA XII inhibitors. PMID:23290254

  20. The impact of Carbonic Anhydrase on the partitioning of leaf and soil CO18O and COS gas exchange across scales

    NASA Astrophysics Data System (ADS)

    Wingate, L.; Wehr, R. A.; Commane, R.; Ogee, J.; Sauze, J.; Jones, S.; Launois, T.; Wohl, S.; Whelan, M.; Meredith, L. K.; Genty, B.; Gimeno, T.; Kesselmeier, J.; Bosc, A.; Cuntz, M.; Munger, J. W.; Nelson, D. D.; Saleska, S. R.; Wofsy, S. C.; Zahniser, M. S.

    2015-12-01

    Photosynthesis (GPP), the largest CO2 flux to the land surface, is currently estimated with considerable uncertainty at between 100-175 Pg C yr-1. More robust estimates of global GPP could be obtained from the atmospheric budgets of other valuable tracers, such as carbonyl sulfide (COS) or the oxygen isotopic composition (δ18O) of atmospheric CO2. However, quantifying GPP using these tracers hinges on a better understanding of how soil micro-organisms modify the atmospheric concentrations of CO18O and COS at large scales. In particular, understanding better the role and activity of the enzyme Carbonic Anhydrase (CA) in soil micro-organisms is critical. We present novel datasets and model simulations demonstrating the progress in the collection of multi-tracer field datasets and how a new generation of multi-tracer land surface models can provide valuable constraints on photosynthesis and respiration across scales.

  1. Anti-tubercular and antioxidant activities of C-glycosyl carbonic anhydrase inhibitors: towards the development of novel chemotherapeutic agents against Mycobacterium tuberculosis.

    PubMed

    Zaro, María J; Bortolotti, Ana; Riafrecha, Leonardo E; Concellón, Analía; Morbidoni, Héctor R; Colinas, Pedro A

    2016-12-01

    During the treatment of tuberculosis infection, oxidative stress due to anti-tubercular drugs may result in tissue inflammation. It was suggested that treatment with antioxidant drugs could be beneficial as an adjunct to anti-tuberculosis drug therapy. Recently our group has shown that several C-glycosides are inhibitors of Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1). In an effort to develop novel chemotherapeutic agents against tuberculosis, the anti-tubercular and antioxidant activities of a series of C-glycosides containing the phenol or the methoxyaryl moiety were studied. Many compounds showed inhibition of growth of M. tuberculosis H37Rv strain and good antioxidant ability. A glycomimetic incorporating the 3-hydroxyphenyl moiety showed the best activity profile and therefore this functionality represents lead for the development of novel anti-tubercular agents with dual mechanisms of action.

  2. Sulfonamides incorporating fluorine and 1,3,5-triazine moieties are effective inhibitors of three β-class carbonic anhydrases from Mycobacterium tuberculosis.

    PubMed

    Ceruso, Mariangela; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2014-10-01

    A new series of fluorine containing 1,3,5-triazinyl sulfonamide derivatives obtained from cyanuric fluoride, sulfanilamide/4-aminoethylbenzenesulfonamide followed and incorporating also amin0, amino alcohol and amino acid moieties have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were efficiently inhibited by these sulfonamides with KI values in the nanomolar or submicromolar range, depending on the substitution of one or both fluorine atoms at the 1,3,5-triazine ring. As some of these enzymes are crucial for the life cycle of this bacterium, the class of β-CA inhibitors reported in this study may lead to antimycobacterial agents with a different mechanism of action compared to the clinically used such drugs for which the pathogen developed extensive drug resistance.

  3. Characterization, bioinformatic analysis and dithiocarbamate inhibition studies of two new α-carbonic anhydrases, CAH1 and CAH2, from the fruit fly Drosophila melanogaster.

    PubMed

    Syrjänen, Leo; Tolvanen, Martti E E; Hilvo, Mika; Vullo, Daniela; Carta, Fabrizio; Supuran, Claudiu T; Parkkila, Seppo

    2013-03-15

    Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30kDa and 28kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO2 hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range. PMID:22989910

  4. Glutamine Induces the N-Dependent Accumulation of mRNAs Encoding Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaf Tissue 1

    PubMed Central

    Sugiharto, Bambang; Suzuki, Iwane; Burnell, James N.; Sugiyama, Tatsuo

    1992-01-01

    We have used detached leaves to study the N-dependent control of expression of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) genes in maize (Zea mays L. cv Golden Cross Bantam T51). Following supplementation with an N-source and zeatin, PEPC and CA mRNA levels increased in leaves detached from N-deficient maize plants. Addition of methionine sulfoximine (MSX), a specific inhibitor of glutamine synthetase, inhibited the nitrate-dependent increase of PEPC and CA mRNA but did not affect the glutamine-dependent increase of PEPC and CA mRNA levels. Glutamine levels in detached maize leaves treated with various N sources in the presence or absence of MSX correlated with the levels of PEPC and CA mRNA. We conclude that glutamine is the most likely effector for controlling the N-dependent expression of PEPC and CA in maize plants. PMID:16653241

  5. Family-wide expression characterization of Arabidopsis beta-carbonic anhydrase genes using qRT-PCR and Promoter::GUS fusions.

    PubMed

    Wang, Meng; Zhang, Qiong; Liu, Fang-Chun; Xie, Wei-Fa; Wang, Guang-Dong; Wang, Jun; Gao, Qing-Hua; Duan, Ke

    2014-02-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes found throughout the phylogenetic tree. The β-class carbonic anhydrases (β-CAs) are the predominating class of CAs in plants. Growing evidence underscores the importance of β-CAs in plant immunity and environmental adaptation in addition to their roles in photosynthesis. However, many fundamental problems in Arabidopsis βCAs expression remain unsolved. Here we examined the transcript abundance of AtβCAs in different tissues of Arabidopsis thaliana, and the accumulation of mRNA in response to CO2 and darkness. Histochemical analysis was performed to study the promoter activity of AtβCAs during post-germination seedling growth and in mature plants. All six members of the AtβCA subfamily showed a response to changed CO2 level and darkness, but each member showed a specific dynamic pattern. Although expression of each AtβCA was unique, in general most AtβCAs were synchronously expressed in green leaves since 5 days after germination until flowering. AtβCA1 and AtβCA2 were most highly expressed in leaves but AtβCA2 displayed weaker expression in roots. The level of AtβCA3 transcripts was highest in flowers, while AtβCA5 was most widely expressed and might be involved in more processes than other members. AtβCA6 was unique for increased expression in darkness and no expression in either the anther or pistil. The present study provides useful information for further functional investigation.

  6. Immunocytochemical localization of V-H(+) -ATPase, Na(+) /K(+) -ATPase, and carbonic anhydrase in gill lamellae of adult freshwater euryhaline shrimp Macrobrachium acanthurus (Decapoda, Palaemonidae).

    PubMed

    Maraschi, Anieli Cristina; Freire, Carolina Arruda; Prodocimo, Viviane

    2015-08-01

    Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps.

  7. Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP–WT1–TBL1 axis

    PubMed Central

    Zhang, Jingwan; Tsoi, Ho; Li, Xiaoxing; Wang, Hua; Gao, Jing; Wang, Kunning; Go, Minnie YY; Ng, Siew C; Chan, Francis KL; Sung, Joseph JY; Yu, Jun

    2016-01-01

    Objective We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. Design The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. Results CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of β-catenin. CA4 interacted with Wilms’ tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms’ tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin β-like protein 1 (TBL1) and the degradation of β-catenin. Conclusions CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP–WT1–TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC. PMID:26071132

  8. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

    PubMed

    Al-Haideri, Halah; White, Michael A; Kelly, David J

    2016-02-01

    Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni. PMID:26470757

  9. The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata.

    PubMed

    Miyamoto, Hiroshi; Miyoshi, Fumiko; Kohno, Jun

    2005-03-01

    Signals and organic matrix proteins secreted from the mantle are critical for the development of shells in molluscs. Nacrein, which is composed of a carbonic anhydrase domain and a Gly-X-Asn repeat domain, is one of the organic matrix proteins that accumulates in shells. In situ hybridization revealed that nacrein was expressed in the outer epithelial cells of the mantle of the pearl oyster Pinctada fucata. The recombinant nacrein protein inhibited the precipitation of calcium carbonate from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the shells of molluscs. Because deletion of the Gly-X-Asn repeat domain of nacrein had a significant effect on the ability of nacrein to inhibit the precipitation of calcium carbonate, it is conceivable that the repeat domain has a primary role in the inhibitory function of nacrein in shell formation. Together these studies suggest that nacrein functions as a negative regulator in calcification in the extrapallial space between the shell and the mantle by inhibiting the precipitation of CaCO3.

  10. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

    PubMed

    Al-Haideri, Halah; White, Michael A; Kelly, David J

    2016-02-01

    Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.

  11. Investigating the adduct formation of organic mercury species with carbonic anhydrase and hemoglobin from human red blood cell hemolysate by means of LC/ESI-TOF-MS and LC/ICP-MS.

    PubMed

    Hogeback, Jens; Schwarzer, Miriam; Wehe, Christoph A; Sperling, Michael; Karst, Uwe

    2016-01-01

    The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.

  12. High Glucose-Induced Mitochondrial Respiration and Reactive Oxygen Species in Mouse Cerebral Pericytes is Reversed by Pharmacological Inhibition of Mitochondrial Carbonic Anhydrases: Implications for Cerebral Microvascular Disease in Diabetes

    PubMed Central

    Shah, Gul N.; Morofuji, Yoichi; Banks, William A.; Price, Tulin O.

    2013-01-01

    Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood-brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration. PMID:24076121

  13. High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes.

    PubMed

    Shah, Gul N; Morofuji, Yoichi; Banks, William A; Price, Tulin O

    2013-10-18

    Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood-brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.

  14. Kinetic isotope effects for concerted multiple proton transfer: a direct dynamics study of an active-site model of carbonic anhydrase II.

    PubMed

    Smedarchina, Zorka; Siebrand, Willem; Fernández-Ramos, Antonio; Cui, Qiang

    2003-01-01

    The rate constant of the reaction catalyzed by the enzyme carbonic anhydrase II, which removes carbon dioxide from body fluids, is calculated for a model of the active site. The rate-determining step is proton transfer from a zinc-bound water molecule to a histidine residue via a bridge of two or more water molecules. The structure of the active site is known from X-ray studies except for the number and location of the water molecules. Model calculations are reported for a system of 58 atoms including a four-coordinated zinc ion connected to a methylimidazole molecule by a chain of two waters, constrained to reproduce the size of the active site. The structure and vibrational force field are calculated by an approximate density functional treatment of the proton-transfer step at the Self-Consistent-Charge Density Functional Tight Binding (SCC-DFTB) level. A single transition state is found indicating concerted triple proton transfer. Direct-dynamics calculations for proton and deuteron transfer and combinations thereof, based on the Approximate Instanton Method and on Variational Transition State Theory with Tunneling Corrections, are in fair agreement and yield rates that are considerably higher and kinetic isotope effects (KIEs) that are somewhat higher than experiment. Classical rate constants obtained from Transition State Theory are smaller than the quantum values but the corresponding KIEs are five times larger. For multiple proton transfer along water bridges classical KIEs are shown to be generally larger than quantum KIEs, which invalidates the standard method to distinguish tunneling and over-barrier transfer. In the present case, a three-way comparison of classical and quantum results with the observed data is necessary to conclude that proton transfer along the bridge proceeds by tunneling. The results suggest that the two-water bridge is present in low concentrations but makes a substantial contribution to proton transport because of its high

  15. A Limited Role for Carbonic Anhydrase in C4 Photosynthesis as Revealed by a ca1ca2 Double Mutant in Maize1[W][OPEN

    PubMed Central

    Studer, Anthony J.; Gandin, Anthony; Kolbe, Allison R.; Wang, Lin; Cousins, Asaph B.; Brutnell, Thomas P.

    2014-01-01

    Carbonic anhydrase (CA) catalyzes the first biochemical step of the carbon-concentrating mechanism of C4 plants, and in C4 monocots it has been suggested that CA activity is near limiting for photosynthesis. Here, we test this hypothesis through the characterization of transposon-induced mutant alleles of Ca1 and Ca2 in maize (Zea mays). These two isoforms account for more than 85% of the CA transcript pool. A significant change in isotopic discrimination is observed in mutant plants, which have as little as 3% of wild-type CA activity, but surprisingly, photosynthesis is not reduced under current or elevated CO2 partial pressure (pCO2). However, growth and rates of photosynthesis under subambient pCO2 are significantly impaired in the mutants. These findings suggest that, while CA is not limiting for C4 photosynthesis in maize at current pCO2, it likely maintains high rates of photosynthesis when CO2 availability is reduced. Current atmospheric CO2 levels now exceed 400 ppm (approximately 40.53 Pa) and contrast with the low-pCO2 conditions under which C4 plants expanded their range approximately 10 million years ago, when the global atmospheric CO2 was below 300 ppm (approximately 30.4 Pa). Thus, as CO2 levels continue to rise, selective pressures for high levels of CA may be limited to arid climates where stomatal closure reduces CO2 availability to the leaf. PMID:24706552

  16. Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein.

    PubMed

    Marie, Benjamin; Luquet, Gilles; Bédouet, Laurent; Milet, Christian; Guichard, Nathalie; Medakovic, Davorin; Marin, Frédéric

    2008-10-13

    The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.