Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. II. Inheritance of red cell carbonic anhydrase levels in different carbonic anhydrase I genotypes of the pig-tailed macaque,Macaca nemestrina

The inheritance of red blood cell levels of carbonic anhydrase isozymes (CA I and CA II) has been studied in different carbonic anhydrase I genotypes of the pig-tailed macaque, Macaca nemestrina. Quantitation of CA I isozymes in a series of animals indicates that the total CA I concentration is the sum of the average effects of each CA I structural allele and that the average effects are independent of the various allelic combinations. The relative average effects were 0.32:0.95:1.0 for the CA I ^a, CA I^b, and CA I ^c structural genes, respectively. It is also demonstrated that the level of CA II is related to the CA I genotypes. Multiple regression analysis demonstrated that each dose of CA I-deficiency gene present decreased the CA II concentration by approximately 30%, with this decrease in CA II level being solely related to the dose of CA I-deficiency gene and not to the level of CA I. The CA I-deficient animals produce CA I products that are similar to the common CA Ia, CA Ib, CA Ic electrophoretic types. Limited mating data indicate that the CA I components in CA I-deficient animals are inherited codominantly.Supported by U.S. Public Health Service Research Grant GM-15419.This report is a portion of a dissertation submitted to the University of Michigan in partial fulfillment of the requirements for the Doctor of Philosophy degree.U.S. Public Health Service Predoctoral Trainee (GM-71-14).     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. I. The radioimmunosorbent assay

A radioimmunosorbent technique is described which is capable of independently detecting both isozymes of carbonic anhydrase, CA I and CA II, in concentrations as low as 1 ng/ml. The technique is used to quantitate the different electrophoretic variants of red cell CA I as well as levels of CA II in the pig-tailed macaque, Macaca nemestrina.Supported by U.S. Public Health Service research grant GM-15419.U.S. Public Health Service Predoctoral Trainee (GM-71-14).     

Quantitative genetic variation in carbonic anhydrase isozymes from tissues of the pig-tailed macaque,Macaca nemestrina

Two isozymes of carbonic anhydrase (CA I and CA II) were quantified by a radio-immunoassay in 10 different tissues of the pig-tailed macaque. There were clearly differences in relative amounts of the two isozymes, indicating a differential regulation of these two different gene products. An inherited deficiency variant reduced red cell CA I and CA II 5000-fold and 2.7-fold, respectively. In nine other tissues, CA I was reduced from approximately twofold to 110-fold, and CA II was essentially unchanged. The CA I in deficient red cells was immunochemically and electrophoretically identical to common electrophoretic variants of CA I in the pig-tailed macaque and was enzymatically active.This work was part of a doctoral dissertation submitted in partial fulfillment of the Doctor of Philosophy degree in the Horace H. Rackham School of Graduate Studies at The University of Michigan. Supported by NIH training grant 5-T01-GM-71-11 and NIH research grant GM-15419.     

Genetic variation and evolution in the red cell carbonic anhydrase isozymes of macaque monkeys

The electrophoretic phenotypes of the two isozymes of red cell carbonic anhydrase, CA I and CA II, are described in nine species of macaque monkeys from southeast Asia and Japan. Twelve phenotypes of CA I, apparently under the control of seven alleles, and five phenotypes of CA II, under the control of three alleles, were found in the different macaque populations studied. Extensive electrophoretic polymorphisms of CA I were found in three species (Macaca nemestrina, Macaca speciosa, and Macaca fuscata), and polymorphisms at the CA II locus were found in Macaca irus, Macaca mulatta, and M. nemestrina. In addition to the electrophoretic polymorphisms at the CA I locus in M. nemestrina, an inherited deficiency of CA I was also discovered in which approximately 30% of the individuals in all populations of M. nemestrina tested showed the deficient phenotype. Although the recessive gene controlling this deficiency appears to be an allele of the CA I locus, it is postulated that the CA I deficiency could also be under the control of a closely linked gene. The comparative data on the extent of genetic variation observed in the two isozymes of red cell carbonic anhydrase in macaques appear to support the concept that CA I has evolved more rapidly than CA II in mammals.Supported by USPHS grant GM-15419 and NSF grants GF-253, GB-7426, and GB-15060 of the U.S.-Japan Cooperative Science and Systemic Biology Programs.     

The cytoplasmic isozymes of carbonic anhydrase in the gastrointestinal tract of reindeer

The cytoplasmic isozymes of carbonic anhydrase: CA I and CA II, in the gastrointestinal tract tissues of reindeer were identified by electrophoresis and substrate-inhibitory tests. The study of the tissue distribution and composition of the cytoplasmic isozymes has shown, that isozyme CA II is found in the forestomachs and the colon mucosal tissue, whereas isozyme CA II predominates in the abomasum and small intestinal mucosa tissue.     

Genetic control of quantitative variation in carbonic anhydrase isozymes of mammals. I. The concentration within single erythrocytes of the mouse

The levels of the carbonic anhydrase isozymes (CA and CA II) in single erythrocytes of DBA/2J mice were assayed by measuring the specific immunofluorescence of CA I and CA II with a microspectrofluorometric technique. Measurements of 100 randomly selected cells showed a range (in relative fluorescence units) of 21-52 (mean 31.3 +/- 7.5) for CA I and 30-80 (mean 45.6 +/- 10.7) for CA II. The CA II/CA I ratio of the means obtained by the single-cell fluorescence assay was similar to the ratios obtained for the two isozymes from hemolysates of DBA/2J mice using an immunodiffusion assay. The influence of cell age on the variation in carbonic anhydrase levels was determined by separating red cells into several fractions by a gravity sedimentation procedure. The younger cells showed higher levels of CA I and CA II than the older cells; however, the extensive overlap in the variability between the cells from the different fractions indicated that although cell age was contributing to the overall heterogeneity, its influence was not pronounced.     

Novel carbonic anhydrase isozymes I, II and IV activators incorporating sulfonyl-histamino moieties.

Sulfonylamido(ureido) derivatives of histamine were synthesized by an original procedure in order to obtain tight-binding activators of the zinc enzyme carbonic anhydrase (CA), exploiting the binding energy of the alkyl/arylsulfonyl moieties with amino acid residues at the entrance of the active site. In contrast to the lead molecule, histamine, the new derivatives possessed higher affinity for three different CA isozymes, as evidenced by compairing the affinity constants of these compounds for isozyme CA II.     

Genetic aspects of quantitative variation in the carbonic anhydrases of the pig-tailed macaque,Macaca nemestrina. I. Response to thyroxine

Effects of thyroxine on incorporation of l-serine-C¹⁴ into four carbonic anhydrase isozymes (CA II, CA Ia, CA Ib, CA Ic) and hemoglobin were quantified in reticulocytes of Macaca nemestrina in vitro. Response to thyroxine differed significantly between CA Ia and two allelic variants (CA Ib and CA Ic) and the nonallelic isozyme (CA II). The effects of thyroxine on serine incorporation into hemoglobin and three of the carbonic anhydrase isozymes were shown to be nonlinear with thyroxine concentration.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 degrees C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Activation of mammalian skeletal-muscle carbonic anhydrase III by arginine modification

Purified carbonic anhydrase isozymes I, II, and III (CA I, CA II, CA III) from various sources were treated with 2,3-butanedione and their bicarbonate dehydration reactions followed. The specific activities of human and bovine CA I and CA II and chicken CA III were not affected by the butanedione treatment, whereas the activities of human, gorilla, and bovine CA III were rapidly activated. These findings suggest that one, or both, of the two arginyl residues which appear to be unique to the active sites of the mammalian CA III isozymes are modified by butanedione.     

Carbonic anhydrase inhibitors. Inhibition of the cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-thiadiazole- and 1,2,4-triazole-thiols

A series of heterocyclic mercaptans incorporating 1,3,4-thiadiazole- and 1,2,4-triazole rings have been prepared and assayed for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II, and the transmembrane, tumor-associated hCA IX. Against hCA I the investigated thiols showed inhibition constants in the range of 97 nM to 548 microM, against hCA II in the range of 7.9-618 microM, and against hCA IX in the range of 9.3-772 microM. Thiadiazoles were generally more active than triazoles against all investigated isozymes. Generally, the best inhibitors were the simple derivative 5-amino-1,3,4-thiadiazole-2-thiol and its N-acetylated derivative, which were anyhow at least two orders of magnitude less effective inhibitors when compared to the corresponding sulfonamides, acetazolamide, and its deacetylated derivative. An exception was constituted by 5-(2-pyridylcarboxamido)-1,3,4-thiadiazole-2-thiol, which is the first hCA I-selective inhibitor ever reported, possessing an inhibition constant of 97 nM against isozyme I, and being a 105 times less effective hCA II inhibitor, and 3154 times less effective hCA IX inhibitor. Thus, the thiol moiety may lead to effective CA inhibitors targeting isozyme I, whereas it is a less effective zinc-binding function for the design of CA II and CA IX inhibitors over the sulfonamide group.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

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.     

Inhibition of human carbonic anhydrase isozymes I, II and VI with a series of bisphenol, methoxy and bromophenol compounds

Carbonic anhydrase inhibitors (CAI) are valuable molecules as they have several therapeutic applications, including anti-glaucoma activity. In this study, inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I, II and VI with a series of bisphenol and bromophenol derivatives was investigated. Molecular docking studies of a set of such inhibitors within CA I and II were also performed. K(I) values of the molecules 2-9 were in the range of 10.025-892.109 μM for hCA I, 1.437-59.107 μM for hCA II and 11.143-919.182 μM for hCA VI, respectively. Reported inhibitory activities of molecules 2-9 will assist in better understanding of structure-activity relationship studies of CAI.     

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.     

Carbonic anhydrase inhibitors. Selective inhibition of human tumor-associated isozymes IX and XII and cytosolic isozymes I and II with some substituted-2-mercapto-benzenesulfonamides

A series of 2-mercapto-substituted-benzenesulfonamides has been prepared by a unique two-step procedure starting from the corresponding 2-chloro-substituted benzenesulfonamides. Compounds bearing an unsubstituted mercapto group and the corresponding S-benzoyl derivatives were investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), i.e., the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor associated isozymes CA IX and XII. These derivatives were medium potency hCA I inhibitors (K(I)s in the range of 1.5-5.7 microM), two derivatives were strong hCA II inhibitors (K(I)s in the range of 15-16 nM), whereas the others showed weak activity. These compounds inhibited hCA IX with inhibition constants in the range 160-1950 nM and hCA XII with inhibition constants in the range 1.2-413 nM. Some of these derivatives showed a certain degree of selectivity for inhibition of the tumor-associated over the cytosolic isoforms, being thus interesting leads for the development of potentially novel applications in the management of hypoxic tumors which overexpress CA IX and XII.