Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Characterization of a high activity carbonic anhydrase isozyme purified from erythrocytes of Salmo gairdneri

1. A single, high specific activity carbonic anhydrase (CA) isozyme was present in erythrocytes of the teleostean species Salmo gairdneri (rainbow trout). 2. Purification of trout CA to homogeneity was accomplished using chloroform ethanol extraction, Sephadex G-75 gel filtration, and DEAE Bio-Gel anion exchange chromatography. 3. Trout CA was a zinc metalloenzyme of mol. wt 28,300 and pI9.3. 4. Amino acid analysis indicated the presence of 6 half-cystine residues per enzyme molecule, and the presence of a sulfhydryl reducing agent was required to maintain full activity in vitro. 5. Sulfhydryl modification with both N-ethylmaleimide and acrylonitrile indicated the presence of 3 reactive sulfhydryl groups per CA molecule. Modification of those groups had no direct effect on enzyme activity, but modified CA was no longer subject to inactivation by oxidizing conditions.     

Characterization of carbonic anhydrase from Neisseria gonorrhoeae.

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO(2) hydration and the similar behaviour of the kinetics of (18)O exchange between CO(2) and water at chemical equilibrium. The pH profile of the turnover number, k(cat), can be described as a titration curve with an exceptionally high maximal value of 1.7 x 10(6) s(-1) at alkaline pH and a pK(a) of 7.2. At pH 9, k(cat) is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio k(cat)/K(m) is dependent on two ionizations with pK(a) values of 6.4 and 8.2. However, an (18)O-exchange assay identified only one ionizable group in the pH profile of k(cat)/K(m) with an apparent pK(a) of 6.5. The results of a kinetic analysis of a His66-->Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar K(i) values for the inhibitors NCO(-), SCN(-) and N(3)(-) as HCA II, while CN(-) and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO(-), SCN(-), CN(-) and N(3)(-) resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO(2) hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A(292)/A(260) ratio was not affected by the presence of TCEP, and a structural transition at 2.8--2.9 M GdnHCl was observed.     

Inhibitory effects of some drugs on carbonic anhydrase enzyme purified from Kangal Akkaraman sheep in Sivas,Turkey

In this study, carbonic anhydrase (CA) enzyme was purified and characterized from blood samples of Kangal Akkaraman sheep and inhibitory properties on certain antibiotics were examined. CA purification was composed of preparation of the hemolysate and conducting the Sepharose‐4B‐tyrosine‐sulfanilamide affinity gel chromatography in having specific activity of 11626 EU mg^?1, yield of 14.40%, and 242.76‐fold purification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed to assess the enzyme purity and a single band was observed. Some antibiotics were exhibited in vitro inhibition on the CA activity. IC₅₀ values of these inhibitors were calculated by plotting activity percentage. IC₅₀ values of certain drugs (dexamethasone; caffeine; metamizole sodium; tetramisol; ceftiofur HCl; ivermectin; tavilin 50; penokain G; neosym; and sulfamezathine) were found as 0.38, 8.24, 285.53, 114.77, 5.33, 2.76, 27.58, 213.50, 208.28, and 36.60 μM, respectively. K_i values of different drugs on Kangal Akkaraman sheep blood CA activity were found in the range of 0.21 ± 0.038–266.64 ± 37.11 μM.     

A radioimmunoassay for quantifying carbonic anhydrase isozymes in crude lysates

A radioimmunoassay was developed for quantifying each of the two genetically distinct forms of primate carbonic anhydrase, carbonic anhydrases I and II, in unpurified lysates. Under the given experimental conditions, the assay is capable of detecting a minimum of 0.025 g of carbonic anhydrase I and 0.005 g of carbonic anhydrase II. There is approximately 98% reproducibility upon repeated assays of a given hemolysate.Supported by NIH training grant 5-TO1-GM-71-11 and NIH research grant 1-PO1-GM-15419-02.NIH postdoctoral trainee.     

Characterization and inhibition effects of some metal ions on carbonic anhydrase enzyme from Kangal Akkaraman sheep

In this work, the carbonic anhydrase (CA) enzyme was purified from Kangal Akkaraman sheep in Sivas, Turkey with specific activity value of 6681.57 EU/mg and yield of 14.90% with using affinity column chromatography. For designating the subunit molecular mass and enzyme purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis method was used and single band for this procedure was obtained. The molecular mass of CA enzyme was found as 28.89 kDa. In this study, the optimum temperature and optimum pH were obtained from 30 and 7.5. V_max and K_m values for p‐nitrophenylacetate substrate of the CA were determined from Lineweaver–Burk graphs. Additionally, the inhibitory results of diverse heavy metal ions (Hg⁺, Fe²⁺, Pb²⁺, Co²⁺, Ag⁺, and Cu²⁺) on sheep were studied. Indeed, CA enzyme activities of Kangal sheep were investigated with using esterase procedure under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC₅₀) and K_i values were obtained.     

Characterization of human carbonic anhydrase III from skeletal muscle

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.     

Thermal stability of carbonic anhydrase immobilized within polyurethane foam

Thermal stability of carbonic anhydrase (CA) immobilized within polyurethane (PU) foam was investigated. The catalytic activity of the enzyme was estimated by using p‐nitrophenyl acetate (p‐NPA) as the substrate in tris buffer containing 10% acetonitrile. The immobilized CA was stable during the repeatable washings and stability tests over 45 days stored in tris buffer at ambient conditions indicating that the CA was covalently attached to the polyurethane (PU) foam by crosslinking. The immobilized CA was found to be 98% stable below 50°C, whereas a drastic decrease was seen at temperatures between 50 and 60°C. The optimum temperature for the immobilized CA was found to be 45°C and it lost its activity completely at 60°C. Thermal deactivation energies for the free and immobilized CA were estimated to be 29 and 86 kcal/mol, respectively. The association of unfolded CA with the polymeric backbone chains of the PU foam was also addressed. It was concluded that the immobilized CA was highly stable at temperatures less than 50°C and could be used in biomimetic CO₂ sequestration processes. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.     

Invertebrate red blood cell carbonic anhydrase

This is the first report documenting the presence of carbonic anhydrase (CA) for any invertebrate red cells. CA activity was measured in plasma, hemolysates of blood cells, and in hemolymph of selected species of invertebrates. Annelid red blood cells (RBC) and sipunculid pink blood cells both possessed significant levels of CA activity. Molluscan RBC, on the other hand, lacked CA activity. The distribution appears to have fallen along phylogenetic lines, with CA being present only in blood cells of the two more closely related groups. However, the presence of extracellular CA was confirmed in oyster hemolymph. Oyster hemolymph CA showed a similar affinity (Ki) for the sulfonamide inhibitors acetazolamide and ethoxzolamide, as did the vertebrate RBC CA II isozyme, supporting the idea that this isozyme could be the ancestral form of the enzyme.     

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration

Diversified group of bacteria were screened for carbonic anhydrase (CA) activity. Significant CA activity was found in crude enzyme extracts of Enterobacter and Aeromonas isolates while minimal or negligible CA activity was observed in case of Shigella and Klebsiella spp. Optimization and characterization study of potent CA producing isolates revealed that the maximum enzyme activity of 3.86 EU/ml was observed in E. taylorae and the optimum pH range for enzyme stability was found to be 7.5–9.0 along with an optimum temperature range of 35–50 °C. The molecular mass of CA was 29-kDa indicating α-type with periplasmic and cytosolic location. Present investigation for the first time reports CA in diversified genus and optimized parameters for enhanced production of CA in Enterobacter sp. & Aeromonas sp. from fresh water bodies that inturn lay down grounds for exploitation of CA from E. taylorae as an efficient catalyst for CO₂ sequestration within a bioreactor.     

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.     

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

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.     

Inhibitory effect of thyroxine on carbonic anhydrase B isozyme biosynthesis in rabbit reticulocyte lysates.

Biosynthesis of rabbit red cell carbonic anhydrase isozyme B and C was demonstrated in reticulocyte cell-free lysates by the specific immunoprecipitin reaction. Using this homologous protein synthesis system, it was found that 10^?5 to 10^?7 M thyroxine preferentially inhibited the synthesis of carbonic anhydrase B isozyme without affecting that of C isozyme. These results suggested that this inhibitory action of the protein synthesis by thyroxine may be responsible for the decreased level of the B type isozymes in human hyperthyroidism or experimental hyperthyroidism of rabbits.