High-activity Form Research Articles

Elasmobranch Carbonic Anhydrase: PURIFICATION AND PROPERTIES OF THE ENZYME FROM TWO SPECIES OF SHARK

Homogeneous carbonic anhydrase has been prepared from erythrocytes of the bull and tiger shark. Molecular and equivalence weight determinations indicate molecular weights in the range of 36,000 to 40,000 depending on the method employed. Amino acid compositions are similar to those reported for mammalian carbonic anhydrases with several notable exceptions. The bull and tiger shark enzymes contain ∼25 and 18 half-cystine residues per molecule, respectively. These residues are apparently present as disulfide cross-links, since neither the native nor denatured molecules react with sulfhydryl reagents, but do react after reduction with mercaptoethanol. There are high contents of glutamic and aspartic acid compatible with the very acidic nature of these proteins. The isoelectric point of the bull shark enzyme is ∼pH 4.5. The shark enzymes catalyze both the hydration of CO2 and the hydrolysis of certain aromatic esters. Maximal catalytic rates are comparable to the high activity forms of mammalian carbonic anhydrases. Aromatic sulfonamides are powerful inhibitors. Circular dichroic spectra in the ultraviolet region indicate the presence of many structural features similar to those found in the mammalian enzymes. The shark enzymes contain 1 g atom of zinc per mole of enzyme. A stable metal-free, inactive apoenzyme can be prepared which is completely reactivated by Zn(II) or Co(II) ions. The Co(II) derivatives have intense visible absorption maxima at 510, 555, 618, and 645 nm. These spectra, as well as the spectra of sulfonamide complexes of the Co(II) enzymes, are almost identical with those of the Co(II) derivatives of mammalian carbonic anhydrases, and suggest that similar coordination geometries exist at the active centers of the elasmobranch and mammalian enzymes. Since fossil morphological evidence suggests that present day shark species have not evolved much since the Devonian period, this particular type of metal complex would appear to have been a constant feature of carbonic anhydrase over 400 million years of animal evolution.

The Carbon Dioxide Hydration Activity of Carbonic Anhydrase: I. STOP-FLOW KINETIC STUDIES ON THE NATIVE HUMAN ISOENZYMES B AND C

The kinetics of catalysis of CO2 hydration by human carbonic anhydrases B and C (EC 4.2.1.1) has been reinvestigated with use of an improved pH indicator stop-flow approach that was checked by studying the uncatalyzed rate of hydration. The Michaelis-Menten parameters were determined between pH 5.8 and 8.8 in noninhibitory buffers. For both isoenzymes Km was independent of pH, whereas Vmax increased with increasing pH. These results are similar to the findings of others on bovine carbonic anhydrase, but differ from the earlier report of Gibbons and Edsall (J. Biol. Chem., 239, 2539 (1964)) in the finding that Km is pH independent. Despite the differences between the low and high activity forms of carbonic anhydrase, there are close kinetic similarities between them that indicate underlying similarities in active site structure. Imidazole inhibits the B enzyme, apparently competitively, but has no effect on the C enzyme. N-Methyl substitution of imidazole abolishes its inhibitory effect. Nitrous oxide does not inhibit catalysis of CO2 hydration by either of the two human isoenzymes, or by bovine carbonic anhydrase. The infrared absorption studies of Riepe and Wang (J. Biol. Chem., 243, 2779 (1968)) had indicated that N2O and CO2 bind competitively to a site identified as the substrate-binding site. The present kinetic results are interpreted as representing a great specificity of carbonic anhydrase for the binding of its substrate CO2. It is proposed that the enzyme-catalyzed hydration of CO2 requires, not only water activation by a basic group, but also charge neutralization in the transition state by an electron acceptor function. The zinc-bound water in carbonic anhydrase could be involved in both donor and acceptor roles.

Carbonic anhydrase isoenzymes in the erythrocytes and uterus of the rabbit.

1. Two forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from rabbit erythrocytes and two forms from rabbit uterine tissue (endometrium) in the progestational stage of pregnancy (days 6-8 of gestation). Separation of the isoenzymes was achieved by ion-exchange chromatography, preparative polyacrylamide-gel electrophoresis and isoelectric focusing. A comparison was made of the general properties and kinetic behaviour of the purified isoenzymes. 2. Although indistinguishable in terms of molecular weight and zinc content the isoenzymes were very different as catalysts of the hydration of carbon dioxide. The two erythrocyte isoenzymes, found in almost equal amounts, differed more than 100-fold in specific activity. Of the two isoenzymes prepared from either endometrial or entire uterine homogenates one was kinetically indistinguishable from the erythrocyte high-activity form, whereas the other, also possessing high activity, was found only in the endometrial or uterine tissue. Present evidence suggests that the former isoenzyme originated from residual blood contaminating the tissue homogenates, and that a marked rise in the content of the latter isoenzyme accounts for the increase in rabbit endometrial carbonic anhydrase activity that previously has been observed in early pregnancy. 3. Minor forms of the erythrocyte isoenzymes, having a characteristic quantitative and electrophoretic relationship to one another, were occasionally produced during purification. 4. The actions were investigated of the inhibitors acetazolamide (5-acetamido-3,4-diazole-1-thia-2-sulphonamide), 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxyzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was superior to the high-activity form as a catalyst of beta-naphthyl acetate hydrolysis.

Properties of Carbonic Anhydrase Isozymes Isolated from Porcine Erythrocytes

Abstract Two major components of carbonic anhydrase were purified from porcine red cells by column chromatography and electrofocusing techniques. Both forms behaved as single components in sedimentation velocity experiments and during starch gel electrophoresis. The observed molecular weight of both forms was about 3 x 104. On the basis of their specific CO2 hydrase activities and amino acid compositions, these two carbonic anhydrase isozymes were designated as high activity (carbonic anhydrase C) or low activity (carbonic anhydrase B) forms which appear to be homologous to the high and low activity carbonic anhydrases, respectively, of other mammals. When these pig B and C isozymes were compared with the red cell carbonic anhydrases of other ungulates (cattle and horse), several interesting features were observed. In contrast to the electrophoretic gel patterns of the horse B and C isozymes in which the C form is markedly more basic than the B form, the high activity C form of pig was observed to be more acidic than the low activity B form. The tryptic peptide map of bovine carbonic anhydrase appears to be more similar to that of porcine carbonic anhydrase C than to B, indicating that they are probably homologous proteins. Neoprontosil binding by the pig and horse B isozymes give rise to essentially identical spectra in the 425- to 600-mµ region, whereas the C isozymes, from these two sources, generate quite different spectra.

Some Characteristics of Human, Bovine, and Horse Carbonic Anhydrases As Revealed by Inactivation Studies

Two representatives of the low (human Enzyme B and horse Enzyme B) and two representatives of the high (bovine Enzyme B and human Enzyme C) catalytic activity forms of erythrocyte carbonic anhydrase were reacted with bromoacetate and bromoacetazolamide with the aim of elucidating structural differences at their active sites. The dissociation constants of the reversible inhibition of bromoacetate and bromoacetazolamide have been determined. It was found that the constants for bromoacetate showed 25- to 30-fold differences between the low and high activity forms of the enzyme, but only 1.1- to 6-fold differences within one activity type. The dissociation constants of the enzyme-bromoacetazolamide complex were almost the same for the two groups. Several lines of evidence suggest that bromoacetazolamide inactivates both types of enzymes with simultaneous alkylation of a histidine (or histidines) at or near the active site at the 3-nitrogen position. The rate of alkylation is, however, markedly different for the two types. Stoichiometric amounts of this inhibitor partially alkylate a histidine of the high catalytic activity forms, but do not react with the low catalytic activity forms. At higher inhibitor concentrations both types react, but the high activity forms react significantly faster. By contrast, bromoacetate at low concentrations inactivates exclusively the low catalytic activity forms with the carboxymethylation of a histidine at the 3-nitrogen position. Evidence is presented that this reaction for the horse enzyme occurs at or near the active site. At higher inhibitor concentrations the high catalytic activity forms also became inactivated, but this reaction is nonspecific. The half-times of the inactivation by bromoacetate were also determined. These values for the high catalytic activity enzymes were found to be 36 times greater than could be explained on a basis of enzyme-inhibitor complex concentration. It is suggested that a difference in the conformation of the active site, or a steric hindrance brought about by an amino acid side chain sufficiently close to the reactive histidine, would account for the observed differences in the rates of alkylation and inactivation.

35Cl-NMR studies of Co 2+ carbonic anhydrases

35Cl NMR studies of Co 2+ substituted carbonic anhydrase (CA) reveal a difference in the pK of hydrolysis for the high and low specific activity forms of the enzyme in agreement with studies on the zinc enzymes. A time dependence was observed for the reaction of Co 2+ with bovine apo CA but not with human B apo CA. A CN − titration of the Co 2+ CA indicates only one cobalt-chloride binding site. This work indicates that 35Cl NMR can be used to monitor the interaction of Co 2+ ions with proteins.

Ribulose diphosphate carboxylase: III. Altered forms of ribulose diphosphate carboxylase from mutant tomato plants

Ribulose diphosphate carboxylase from many mutant forms of the tomato plant have been studied. Enzymes with altered activity indicative of both high and low specific activity forms have been observed. These enzymes differ in their electrophoretic mobilities and also in their kinetic dependences upon substrate concentrations. Two mutant forms of the enzyme with high specific activity and two with low specific activity have been studied. These ezymes also differ in their ability to bind and to be activated by a protein containing light activating factor.

Carbonic anhydrase isoenzymes in the erythrocytes and dorsolateral prostate of the rat.

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform-ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of beta-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

Purification and Properties of Horse Erythrocyte Carbonic Anhydrases

Abstract Two distinct components of carbonic anhydrase (EC 4.2.1.1) have been purified from horse red cells. In the isolation procedure described, the large excess of hemoglobin was removed by precipitation in ammonium sulfate, and the two carbonic anhydrase proteins were then separated by DEAE-Sephadex chromatography. This separation was facilitated by the remarkably high isoelectric point of the basic component, which appeared to have a net positive charge even at pH 10.0. This basic component, which was designated enzyme C, constituted only 36% of the total carbonic anhydrase protein but had a high specific activity; with the use of the WilburAnderson assay for CO2 hydration, this was in the order of 40,000 units per mg. The major protein, designated enzyme B, had a much lower specific activity, in the order of 4,000 units per mg. The extinction coefficients and s20,w values of the purified B and C proteins were measured, as well as the Km and Vmax values for their hydrolysis of p-nitrophenyl acetate. The amino acid compositions and the optical rotatory dispersion spectra are also reported. The two components of horse carbonic anhydrase described here give a further example of the unusual type of polymorphism already seen with primate carbonic anhydrase. This suggests that it may be a characteristic of the enzyme from the blood of a variety of mammalian species. The salient features seem to be firstly, the presence of two chemically distinct proteins catalyzing the same reaction at different rates, and secondly, the much greater abundance of the less active form. The isolation of two horse enzymes brings to seven the number of carbonic anhydrases now studied in a highly purified state. These are classified as either high specific activity forms (human, rhesus monkey, and horse C enzymes, bovine B enzyme) or low specific activity forms (human, rhesus monkey, and horse B enzymes), and the proteins in each category are then compared in terms of amino acid composition, kinetics, and optical rotatory dispersion.

CARBONIC ANHYDRASE ISOZYMES OF NEONATAL AND ADULT HUMAN AND SOME ANIMAL ERYTHROCYTES.

SummaryIon exchange separations utilizing DEAE‐cellulose are described that are capable of isolating carbonic anhydrases individually or in groups as they occur in bovine, guinea pig, adult human, or neonatal human erythrocytes.Acrylamide gel electrophoresis procedures have been utilized for evaluation of purification processes as well as for demonstration of relative carbonic anhydrase activities of the various enzymes and for estimation of their esterase activities.Guinea pig carbonic anhydrases, similar to those from human erythrocytes, have both high and low specific activity forms, while the bovine enzymes demonstrate no such difference in activity.The pattern of the neonatal resembles that of the major adult human carbonic anhydrases; a reduction in activity, however, is apparent for the principal high activity form of the enzyme. The total recoverable enzyme as well as the measurable activity is lower in neonatal than adult erythrocytes.The active carbonic anhydrases in the three species studied have esterase activities independent of carbonic anhydrase activity but approximately proportional to protein concentration. Inasmuch as these enzymes hydrolyze napthyl acetates but are very inactive toward α‐napthyl propionate, α‐napthyl butyrate, or higher napthyl esters, they may be identified as acetyl esterases. They are, however, not sensitive to eserine.