Richard E. Tashian

Carbonic Anhydrase II Deficiency in 12 Families with the Autosomal Recessive Syndrome of Osteopetrosis with Renal Tubular Acidosis and Cerebral Calcification

Osteopetrosis with renal tubular acidosis and cerebral calcification was identified as a recessively inherited syndrome in 1972. In 1983, we reported a deficiency of carbonic anhydrase II, one of the isozymes of carbonic anhydrase, in three sisters with this disorder. We now describe our study of 18 similarly affected patients with this syndrome in 11 unrelated families of different geographic and ethnic origins. Virtual absence of the carbonic anhydrase II peak on high-performance liquid chromatography, of the esterase and carbon dioxide hydratase activities of carbonic anhydrase II, and of immunoprecipitable isozyme II was demonstrated on extracts of erythrocyte hemolysates from all patients studied. Reduced levels of isozyme II were found in obligate heterozygotes. These observations demonstrate the generality of the findings that we reported earlier in one family and provide further evidence that a deficiency of carbonic anhydrase II is the enzymatic basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. We also summarize the clinical findings in these families, propose mechanisms by which a deficiency of carbonic anhydrase II could produce this metabolic disorder of bone, kidney, and brain, and discuss the clinical evidence for genetic heterogeneity in patients from different kindreds with this inborn error of metabolism.

An improved method for the purification of carbonic anhydrase isozymes by affinity chromatography

Methods for purifying carbonic anhydrase (EC 4.2.1.1.) isozymes, carbonic anhydrase B (or I) and C (or IIL by affinity chromatography have been described by Falkbring et al. (1) and Whitney (2), in which affinity gels are formed by coupling, respectively, p-aminobenzenesulfonamide, and p-(aminomethyl)benzenesulfonamide to Sepharose polysaccharides by means of cyanogen bromide activation. Attempts to repeat these methods in our laboratory were not satisfactory as the reproducibility and yields were variable. It is possible that the susceptibility of the linkage to esterase hydrolysis by carbonic anhydrase could account for this variability. It has been suggested (3) that the linkage given by cyanogen bromide activation is of an ester type, i.e., -C-O--CO--NH--R, where R represents a functional group, and it I was possible to hydrolyse this linkage by heating at about 80°C and pH values between 8 and 12. When purified carbonic anhydrase at pH 9.0 was passed through columns of this type of gel, cleavage of the sulfonamides occurred; and in particular, the cleavage products were readily observed when highly colored azosulfonamides were coupled. Also the total distance of the coupled inhibitor from the gel matrix, given by these methods, does not exceed the depth of the active site cleft (4). Therefore, to overcome these factors, gels were prepared by coupling sulfonamides to CM Sephadex using a water-soluble carbodiimide (5) to form a peptide bond between the carboxyl and amino groups. Two sulfonamide inhibitors were used, p[(2,4-diaminophenyl)azo]benzenesulfonamide (Prontosil), and p-(aminomethyl)benezenesuifonamide. The azosulfonamide coupled gel had the advantages of a higher capacity and a red-colored product that enabled visual estimation of coupling efficiency. For recent reviews of carbonic anhydrase isozymes see Lindskog et ah (6), and Carter (7).

Genetics of the mammalian carbonic anhydrases

Publisher Summary This chapter discusses the genetics of the mammalian carbonic anhydrases. The chapter provides information about various evolutionarily genes coding for enzymes that can surpass the carbonic anhydrase (CA) multigene family with respect to the distributional and functional diversity of their gene products. The CA gene family in mammals consists of eight genes coding for seven CA isozymes (CA I–CA VII) and a CA related protein (CARP). These genes may be expressed in certain cells of virtually all tissues or limited in expression to a single tissue, with the other CA genes ranging in their expression between these two extremes. Considerable chemical and biological information is now accumulated on the CAs regarding their primary and tertiary structures, enzyme kinetics, active site mechanisms, physiological roles, cellular and sub cellular locations, genetics, and evolution. Therefore, the related CA genes appear to be an attractive model for the study of the molecular genetics and evolution of a multigene family. It is possible that both types of carbonic anhydrase may be present in bacteria. The CA found in Neisseria sicca— and probably other bacteria—may be related to the algal and animal CAs, whereas the CA products of the cynT gene of E. coli and the IcfA gene of the cyanobacterium, Synechoccus sp. belong to the β-CA family.

Amino Acid Sequence Evidence on the Phylogeny of Primates and Other Eutherians

The biomolecular approach to systematic and evolutionary biology is in a state of transition. Laboratories that had been determining the amino acid sequences of proteins are now caught up by the excitement of the new recombinant DNA gene cloning and sequencing technology. The possibilities for advancing knowledge in systematic and evolutionary biology by application of this new technology seem almost boundless. It is obvious that knowing the actual nucleotide sequences of genes, rather than having to infer them from the amino acid sequences of encoded proteins, allows more accurate data to be used in figuring out the genealogic relationships of organisms (see Hewett-Emmett et al., this volume, Chapter 9; also Scott and Smith, this volume, Chapter 8). During the transition, while laboratories engaged in studying molecular evolution are retooling in order to engage in nucleotide sequencing, it is worth preparing for the impending flood of these gene sequence data by taking stock of what has already been learned about phylogeny from the substantial body of amino acid sequence data. With that objective in mind, this chapter focuses attention on the phylogeny of the order Primates, both on the subbranching within the order and on the genealogic position of Primates within the subclass Eutheria as well as on the broader pattern of vertebrate branching. We will concentrate on these groups because more species are represented in them by amino acid sequence data than in any other eukaryotic branch.

Testosterone-induced, sulfonamide-resistant carbonic anhydrase isozyme of rat liver is indistinguishable from skeletal muscle carbonic anhydrase III

Three isozymes of carbonic anhydrase, CA I, CA II and CA III, have been characterized from the tissues of a variety of vertebrate species. CA I (low-activity, sulfonamide-sensitive form) has been found mainly in red cells but also occurs in other tissues (e.g., rumen epithelium, caecum, colonic mucosa, pituitary gland, ciliary body), CA II (high-activity, sulfonamide-sensitive form) is found in red blood cells and in a majority of other tissues, and CA III (low-activity, sulfonamideresistant form) has been reported to occur largely in red skeletal muscle; cf. [l-7]. Although all 3 isozymes catalyze the reversible hydration of CO*, hydrolyze certain ester linkages, and are selectively inhibited by heterocyclic sulfonamides such as acetazolamide (Diamox), their relative activities and degrees of sulfonamide inhibition can vary considerably. For example, the CA II isozymes have the highest CO2 hydrase and esterase (toward p-nitrophenyl acetate) activities and the highest affinities for sulfonamides, followed by the CA I isozymes, while the CA III isozymes exhibit markedly lower CO2 hydrase and esterase activities and are only weakly inhibited by sulfonamides [l-4]. Early studies on carbonic anhydrase activity in rat liver homogenates [8,9] showed that its CO* hydrase activity was poorly inhibited by acetazolamide compared to the strong inhibition of rat red cell carbonic anhydrase (presumably CA I and CA II) activities. A partial characterization of a carbonic anhydrase isolated from livers of adult male rats [IO] showed that the specific CO* hydrase activity of this form was -1% that of the high-activity CA II isozyme purified

Origins and Molecular Evolution of the Carbonic Anhydrase Isozymesa

Work on membrane-bound and subcellular forms of CA at the protein level, and the possibility of multiple forms of the mouse CA II gene at the DNA level, indicate that CA may represent an extensive multigene family. A method for classifying newly sequenced CA molecules, or genes encoding them, is discussed. Phylogenetic trees based on the existing sequence data are presented and discussed in terms of gene evolution. The active-site residues of CA II have been more conserved in evolution than those of CA I or CA III. After the gene duplications, CA III and CA I initially evolved more rapidly than CA II. Since the mammalian radiation, the CA II molecule as a whole has been accepting substitutions more frequently than CA I, which in turn is evolving more rapidly than CA III. These findings can be explained if external regions of CA I and CA III have been conserved in evolution owing to interactions with other molecules. Two such regions appear to be residues 18-37 in CA I and 231-250 in CA III. Spinach CA was purified and a small amount of sequence data collected. The difficulty in aligning it with animal CAs suggests that a plant CA may not be suitable to shed light on the active site and character of the ancestral eukaryote CA.

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.

Esterase and hydrase activity of carbonic anhydrase-I from primate erythrocytes☆

Abstract Carboxylic esterase activity toward the acetate and butyrate esters of α- and β-naphthol is associated with two forms of erythrocyte carbonic anhydrase (CA-I, CA-II) in man (Tashian et al. , 1963) and other primate species. Of 22 primate species tested, erythrocyte CA-I from the rhesus macaque ( Macaca mulatta ) and doguera baboon ( Papio doguera ) were among those forms showing high specific esterase activity; in comparison, human and chimpanzee ( Pan troglodytes ) CA-I exhibit moderate esterase activity. This report compares the hydrolase and hydrase activities of partially-purified CA-I from human, chimpanzee, baboon, and rhesus hemolysates.

Carbonic anhydrase (CA)-related proteins (CA-RPs), and transmembrane proteins with CA or CA-RP domains

Members of the a-carbonic anhydrase (α-CA) gene family encode not only proteins that exhibit the characteristic catalytic activity of CA (i.e., the reversible hydration of CO2), but also CA-related proteins that are apparently devoid of this activity. For a recent summary of the activity mechanisms and functions of the mammalian CA isozymes, see Sly and Hu (1995). In amniotes (reptiles, birds, and mammals), the active CA isozymes have been designated, CA I — CA VII, CA IX, CA XII, and CA XIII, and the presumed inactive isoforms, CA-RP VIII, CA-RP X, and CA-RP-XI. In non-amniotes, apparently inactive α-CA-related proteins (CAH-RPs) have been identified in both the nematode, C. elegans, and in the yam (Dioscorea). Additional CA-related proteins are present as transmembrane proteins in several pox viruses, and as the CA-RP N-terminal domains of the extracellular regions of the transmembrane proteins of receptor protein tyrosine phosphatases β and γ (RPTPβ and γ). All of these presumably “acatalytic” CA-related isoforms probably either have no, or greatly diminished, CO2 hydration activity due to the substitution of one or more of the three histidine residues that are required to bind the zinc ion that is essential for efficient CA activity.

Biochemical genetics of carbonic anhydrase.

Carbonic anhydrase (EC 4.2.1.1. carbonate dehydratase) appears to be present in placental mammals as two distinct molecular forms, or isozymes, which are apparently under the control of two closely linked autosomal genes. Next to hemoglobin, carbonic anhydrase is the most abundant protein to be found in human erythrocytes. This feature, together with the easily definable electrophoretic phenotypes of the two isozymes, and the relative ease with which they can be purified from hemolysates, has made the carbonic anhydrase isozyme system a particularly attractive one for the study of genetic variation in humans at the molecular level.