Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland     

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.     

Purification and characterization of carbonic anhydrase from bovine erythrocyte plasma membrane

Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membrane and characterized in this study. For this purpose, the blood taken from young animals was hemolysed, the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was removed from the membrane with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography.The CA obtained from erythrocyte membrane has an esterase activity as well as hydratase activity. The Vmax and Km of the enzyme for the substrate (p-nitrophenyl acetate) are 1.948x10(-3) mM/L x dak, and 3.596 mM, respectively. The purification degree of the enzyme was controlled by SDS-PAGE (3-10), which showed two distinct bands. It was determined that the enzyme had activity within the pH range of 4.5-9.5 and that the optimal pH was 7.5. The temperature at which it showed activity was 20-60 degrees C and optimal temperature was 37 degrees C. Molecular weight of CA was found to be 29844 and 61706 Dalton by gel filtration. On the other hand, sulfanilamide and acetazolamide affected the enzyme.     

Purification and characterization of an extracellular carbonic anhydrase from Pseudomonas fragi

Extracellular carbonic anhydrase was purified from Pseudomonas fragi isolated from CaCO₃ enriched soil samples. The enzyme is induced in presence of CaCO₃ and is envisaged to play an important role in bicarbonate ion transport. The 75% ammonium sulphate dialysate was purified by single step affinity chromatography with 86% yield. It is a trimeric protein having a subunit molecular weight of 31.0 kDa and was stable at pH 7.0–8.5 and temperature 35–45 °C. Lead, mercury and EDTA had an inhibitory effect on CA activity, whereas zinc, iron and cadmium increased it. The presence of esterase activity along with IC₅₀ of sulphonamides and anionic inhibitors indicated that CA from P. fragi belonged to α-class. The CA stability in presence of different salts, as well as in alkaline pH and high temperature makes it a potential candidate to be exploited for biomimetic CO₂ sequestration.     

Purification and characterization of carbonic anhydrase from the saliva of the rat

Carbonic anhydrase purified from the saliva of the rat had kinetic properties identical with those of carbonic anhydrase II from rat red cells, but its molecular properties were distinctly different from the type II isozyme. Kinetic parameters were measured under steady state conditions by stopped-flow spectrophotometry and under equilibrium conditions by an 18O exchange method. The turnover number kcat for hydration of CO2 was 6.5 X 10(4) s-1 and the Michaelis constant was 4.2 mM at pH 7.5 and 25 degrees C, values which are equal to the steady state constants for red cell carbonic anhydrase II from the rat. Inhibition of the salivary isozyme by sulfanilamide (Ki = 3.7 microM) was nearly as efficient as inhibition of the erythrocyte isozyme II (Ki = 1.1 microM). The molecular weight for the salivary isozyme was 46,000 and the isoelectric point was 5.5. Salivary carbonic anhydrase had high mannose oligosaccharide components as measured by concanavalin A binding. The amino acid composition for the salivary isozyme was not similar to rat type II, but it was similar to that reported for membrane-bound carbonic anhydrase from bovine lung (Whitney, P.L., and Briggle, T.V. (1982) J. Biol. Chem. 257, 12056-12059). These observations suggest to us that salivary carbonic anhydrase is a secretory product.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Purification and characterization of human salivary peroxidase

Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.

Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with Ki < 0.1 nM for porcine CA II at pH 7.4. Although the Ki is dependent on the CA isozyme type (CA II < CA IV < CA III approximately CA I), it is relatively insensitive to the species source, as long as it is mammalian. The Ki is pH dependent with log Ki decreasing linearly as the pH decreases, implicating at least one ionizable group with the pKa < or = 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.     

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of F?rster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding.     

Complete amino acid sequence of ovine salivary carbonic anhydrase

The primary structure of the secreted carbonic anhydrase from ovine salivary glands has been determined by automated Edman sequence analysis of peptides generated by cyanogen bromide and tryptic cleavage of the protein and Staphylococcus aureus V8 protease, trypsin, and alpha-chymotrypsin subdigests of the large cyanogen bromide peptides. The enzyme is a single polypeptide chain comprising 307 amino acids and contains two apparent sites of carbohydrate attachment at Asn-50 and Asn-239. The protein contains two half-cystine residues at 25 and 207 which appear to form an intramolecular disulfide bond. Salivary carbonic anhydrase shows 33% sequence identity with the ovine cytoplasmic carbonic anhydrase II enzyme, with residues involved in the active site highly conserved. Compared to the cytoplasmic carbonic anhydrases, the secreted enzyme has a carboxyl-terminal extension of 45 amino acids. This is the first report of the complete amino acid sequence of a secreted carbonic anhydrase (CA VI).     

Purification and properties of carbonic anhydrase from salmon erythrocytes

Carbonic anhydrase (CA) from erythrocytes of the pink salmon, Onchorhyncus gorbushka, was purified using chloroform-ethanol extraction and Sephadex G-75 gel filtration. A single, high specific-activity CA isozyme having a molecular weight of 29,000 was found. The enzyme sedimented as a single boundary at a sedimentation velocity of 2.9S. Amino acid analysis revealed a composition similar to other submammalian CAs with the exception that the cysteine content was low (1 mol cysteine/mol enzyme). Like other submammalian CAs, the presence of a sulfhydryl reducing agent was required to maintain full activity and to prevent structural changes in the enzyme.     

Purification and properties of pig muscle carbonic anhydrase III

Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.     

Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.     

Biochemical characterization of the human carbonic anhydrase variant CA ih Hiroshima

Summary Some biochemical properties of a new red cell human carbonic anhydrase variant, CA Ih Hiroshima, have been determined. Evidence is presented that the amino acid substitution in the Japanese variant is not the same as the previously characterized CA Ic variant from Guam of similar electrophoretic mobility. Based on a comparison with the normal CA I isoenzyme, a proposal for the site of the amino acid substitution is presented.     

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

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 838.89 U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35 °C, 20 mM and 8.5, respectively. The molecular weight determined by SDS–PAGE was found to be 29 kDa. The kinetic parameters, K_M and V_max values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The K_i 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 1.348–69.31 μM.     

Synthesis and characterization of a metal-binding peptide fragment of human carbonic anhydrase B

A 36-amino acid residue peptide containing the presumed metal-binding ligands at the active site of human erythrocyte carbonic anhydrase B was synthesized by the standard solid phase method. The synthetic peptide was purified by ion-exchange chromatography and was homogeneous as judged by cellulose acetate gel electrophoresis. Amino acid analysis, dansylation, C-terminal determination, and four cycles of Edman degradation all gave results consistent with the anticipated sequence. The peptide binds Co(II) with an apparent dissociation constant of about 7 × 10^?5M (uncorrected) but has little, if any, of the catalytic activity of carbonic anhydrase. Possible explanations for the weak binding of the metal ion are discussed along with prospects and strategies for designing polypeptide models of enzymatic catalysts.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.