Carbonic anhydrases

Int. J. Biochem. Vol. 19, No. 2, pp. 101-113, 1987

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REVIEW

CARBONIC ANHYDRASES HAROLD F. DEUTSCH* Max-Planck-Institut fiir Zellbiologie, Rosenhof, D-6802 Ladenburg/Heidelberg, West Germany [Tel. (06203) 5098] (Received 28 May 1986)

INTRODUCTION The isolation of carbonic anhydrase (EC 4.2.1.1.) from erythrocytes by Meldrum and Roughton (1933), the first recognized metalloenzyme, essentially initiated a field of investigation which is receiving ever increasing attention. Three enzymatic forms designated CA-I (or B), CA-II (or C) and CA-Ill (or M) have been described in great detail. Each is a single chain peptide of 259 or 260 residues, having a blocked N-terminal group and containing a single zinc which is requisite for its activity. These isozymes are encoded by distinctly separate genes but since their amino acid sequences show a great deal of homology they appear to have a common evolutionary ancestory. The homologies noted are particularly marked for those amino acids which form the so-called "active center". Variations in these residues form the major basis for attempts to clarify the differences in activity noted for the different type isozymes as well as the effects of chemically induced modifications and the actions of various inhibitors. Other distinct forms of carbonic anhydrase than those noted above, which for the most part appear membrane bound, are being described. It is possible that they may represent products of one or more additional genes. However, it is not certain as yet whether these only partially characterized isozymes are encoded by separate genes but they do not appear to be post-translational modifications of well-known forms. Some of the recent studies in this area will be considered briefly. In the present discussion particular attention will be devoted to the carbonic anhydrase of muscle (CA-Ill) since its relatively recent isolation and recognition as a distinct entity (Register et al., 1978) has focused a great deal of attention of its relationship to the well-known erythrocyte CA-I and CA-II forms. Included in this discussion will be a consideration of ubiquitin, a 76 residue protein possessing some of the enzymatic properties of the carbonic anhydrases and having a distinct sequence homology to CA-Ill. A role for carbonic anhydrase related abnormalities to several disease processes also appears to *This review was compiled during the author's tenure as an Alexander von Humboldt Professor. Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wl 53706, U.S.A. B.C 19/2--A

be emerging and lends further interest to these isozymes.

STRUCTURAL ASPECTS Human erythrocytes were utilized in early studies to isolate the carbonic anhydrase used to obtain the first sequence of the 260 residue low activity form now designated CA-I (Andersson et a l , 1972; Lin and Deutsch, 1973; Giraud et al., 1974) and of the 259 residue high activity CA-II form (Henderson et al., 1973; Lin and Deutsch, 1974). Complete sequences of the CA-I isozyme of cows (Tashian et al., 1980), horses (Jabusch et al., 1980), chimpanzee (Contel et al., 1981) and rhesus macaque (Henriksson et al., 1980) and of the CA-II form from bovine (Sciaky et al., 1976), equine (Jabusch and Deutsch, 1984), ovine (Tanis et al., 1974) and lapine (Ferrell et al., 1978) sources and of CA-Ill from cows (Tashian et al., 1980) and from horses (Wendorff et al., 1985) have been reported. Partial sequences of various other carbonic anhydrases have also been determined (Tashian et al., 1981, 1983). Although the high and the low activity enzymes from erythrocytes of a given species usually show sequence identities of more than 50°/'0, the low and the high activity isozymes of different species show much g~eater homologies. When the sequence data of Tashian et al. (1980) and of Engbert et al. (1985) for bovine CA-Ill are compared with that of the equine enzyme (Wendorff et al., 1985) a homology of 93% is found. The latter isozyme shows only a 55°/'o sequence identity with the CA-I and CA-II forms from the same species (Wendorff et al., 1985). These data point to a common ancestorial gene for these isozymes. The molecular evolution of the three well known isozymes has been recently considered at length by Hewett-Emmett et al. (1984). Even though chicken CA-Ill shows homologies to the muscle carbonic anhydrases of mammals (Tashian et al., 1983), the amino acid compositions of this isozyme from different mammalian species reveal considerable differences as shown by the data of Table 1. The result for the feline enzyme (Sanyal et al., 1982b) probably reflects analytical problems since the presence of only 234 instead of 259 residues are indicated even though the usual molecular weight of 28,500 was obtained by gel electrophoresis. 101

102

HAROLD F. DEUTSCH Table I, Amino acid compositions and some properties of five muscle carbonic anhydrases Amino acids Asp Ash Asx Thr Set Glu Gin GIx Pro Gly Ala Val Met lie Leu Tyr Phe Trp His Lys Arg CySH No. Residues (1%, I cm, 280nm) Isoelectric pH

EquinC

BovinC

18 10 (28) 15 16 13 6 (19) 21 19 16 15 I 9 22 10 12 8 11 18 14 5 259 15.5 8.9

14 13 (27) 12 18 16 5 (21) 20 17 20 13 I 12 23 8 II 8 12 20 II 5 259 20.7 8.5 ~

Porcine'

Lapine"

Feline a

25 13 16

28 11 19

22 9 15

19 18 17 17 15 2 13 25 9 0 8 1l 19 19 5 258 22.2

19 23 19 14 14 2 10 20 9 12 10 11 IS 13 6 260 23.2 8.4U

21 19 18 17 15 3 10 24 8 8 5 8 17 I1 4 (234)

°From sequence data of Wendorff et al. (1985). bFrom sequence data of Tashian et al. (1980) with corrections of Engberg et al. (1985). CPullan and Noltmann (1984). dSanyal et al. (1982). eEngberg et aL (1985). fRegister et al. (1978).

The differences noted for the amino acid analysis of the CA-III isozymes are also reflected in variations in their isoelectric points (see Table 1) even though the equine, bovine and lapine enzymes are all relatively basic. It appears that the pIE of 7.4 for the human (Carter et al., 1979) and of 6.28 for the rat (King et al., 1974) CA-Ill isozymes represent a considerable difference in their charge properties from those of the other species. The relatively small protein ubiquitin, molecular weight 8565, has been found to possess esterase activity for the usual substrates employed in studies with the carbonic anhydrases and to also have a weak CO2 hydratase action that is inhibited by acetazolamide (Matsumoto et al., 1984). Ubiquitin possesses a sequence homology to residues 52-127 of CA-Ill. Although only 14 of ubiquitin's 76 residues are identical to this region of bovine CA-Ill, the average minimal residue base difference in the codons is only 0.4211 compared to an anticipated random difference of 1.4969 (Deutsch, 1984). This indicates that there would be only 1 chance in 106 that the sequence homology of these two proteins could be random. These considerations are complicated due to ubiquitin being coded for by genes which have from six to nine repeats, said repeats differing significantly in their nucleotide sequences but encoding the same amino acids (Ozkaynak et al., 1984; Wiborg et al., 1985; Bond and Schlesinger, 1985; Dworkin-Rastl, 1984; Lund et aL, 1985). Almost all of the sequence data for the carbonic anhydrases reported to date were obtained by the usual Edman degradation method. However, the sequence of murine CA-Ill has been deduced from the sequence of the cDNA coding for this protein (Curtis et al., 1983). It is likely that most sequences

of carbonic anhydrases in the future will be derived from the requisite polynucleotide sequence. In the past several years significant progress has been made toward the isolation of D N A probes for various carbonic anhydrases. These include human CA-III (Lloyd et al., 1985), rabbit CA-I (Konialis et al., 1985; Boyer et al., 1984), murine and human CA-II (Venta et al., 1984) and chicken CA-II (Yoshihara et al., 1984). Concomitant with the sequence studies on the human carbonic anhydrases were X-ray crystallographic investigations which provided the 3-dimensional structures of the human high and low activity forms (Liljas et al., 1972; Kannan et al., 1975). These data are discussed in detail by Nostrand et al. (1974), Kannan (1980), and Kannan and Ramanadham (1984). The results from these studies along with the sequences are being used to elucidate such functions as the principles and mechanisms underlying their enzymatic activities, the mode of action of inhibitors and to correlate the relationships between enzymatic activity changes and natural and chemically induced structural variations. Because of the strong sequence homologies among the different carbonic anhydrases it is assumed that all of them possess similar 3-dimensional structures as those elucidated for the human isozymes. From the results of these investigations it is possible to identify 30 active-site residues of which !6 are invariant. These relate to the structure around the zinc moiety. This metal is always bonded to histidines 94, 96 and 119. A hydrogen-bonded network linked to the zinc-bonded H20 and these histidines, either directly or indirectly, includes residues 29-Ser, 92-Glu, 106-Glu, 107-His, ll7-Glu, 194-Tyr, 19%Thr, 209-Trp and 224-Asn. These residues are

Carbonic anhydrases always invariant (Lindskog, 1982; Lindskog et al., 1984). Other residues lining the active site cavity have been grouped into exposed polar and exposed nonpolar amino acids. Some of these polar type residues such as those at positions 64 and 200, appear to exert modifications of catalytic behavior. All CA-I type isozymes have histidines in these two positions whereas the CA-II types have histidine and usually threonine, respectively. Thus the histidine at position 200 in C A d is a unique difference distinguishing it from the CA-II isozymes. CA-III type isozymes have threonine at residue 200 like CA-II but possess lysine or arginine at residue 64. The presence of arginine at positions 67 and 91 is another significant difference in the active-site residues of CA-III isozymes. The effects of chemical modification of these arginines on the enzymatic activities of CA-III will be considered later. Although ubiquitin possesses a sequence homology to CA-III, it possesses only one of the three histidines linked to zinc in these isozymes, namely residue 68 which in ubiquitin is analogous to residue 119 of CA-III. There is a particularly strong homology in this region since residues 67-70 of ubiquitin are identical to residues 118-121 of CA-III. Ubiquitin does not contain zinc and furthermore, the addition of this metal does not enhance its relatively weak enzymatic activity. Other properties that ubiquitin shares with CA-III will also be considered later, The erythrocyte carbonic anhydrases of various nonmammalian species have also been subjected to study. In these cases the classification of whether an isozyme is CA-I or CA-II "like" is often based on the similarity of various of its kinetic parameters to those of the well known mammalian forms. Isolates from frog (Bundy and Cheng, 1976), turtle (Hall and Shraer, 1979; Tashian et al., 1981) and chicken (Bernstein and Shraer, 1972; Tashian et al., 1980, 1983) indicate that these species possess a CA-II type isozyme. Chicken intestinal extracts also reveal the presence of a carbonic anhydrase that may be of the CA-I type (Holmes, 1977). Chicken erythrocyte carbonic anhydrase although having type II activity shows sulfonamide inhibition of the CA-I type. The turtle also contains a low activity isozyme analogous to mammalian CA-I (Hall and Shraer, 1979; Tashian et al., 1981). Acetazolamide inhibition studies of the hagfish enzyme indicates that it behaves similarly to mammalian CA-II (Carlsson et al., 1980). However, its activity appears to be that of the CA-I form (Carlsson et al., 1980; Maren et al., 1980) while the enzyme from sheepshead erythroyctes has an inhibition pattern similar to mammalian CA-II. The turnover number of this teleost isozyme is also closer to that of the low activity mammalian forms (Sanyal et al., 1982a). The erythrocyte carbonic anhydrases from the bull and tiger sharks have molecular weights higher than the mammalian forms and have activities comparable to those of CA-II (Maynard and Coleman, 1971). Plant carbonic anhydrases are generally characterized by their higher molecular weights but appear to be composed of subunits of about 30,000 and like CA-III contain more cysteine than the erythrocyte forms. Even though their amino acid compositions resemble those of the latter isozymes, insufficient

103

data, such as sequence analyses, is available to establish the structural relationships between the plant and various animal forms of these enzymes. Data pertinent to the plant carbonic anhydrases have been summarized by Graham et al. (1984). TISSUE

BOUND

FORMS

The plasma membranes and organelles of a variety of cells have been shown to possess carbonic anhydrase activities that appear to be integral parts of these structures. They most often have been demonstrated by histochemical techniques such as the method of Hansson (1967) or variations of this approach (Ridderstrhle, 1976; Sugai and Ito, 1980; L6nnerholm, 1980). Immunocytochemical techniques using antibodies to one of the three soluble enzymes, CAd, CA-II or CA-III, permits definition of which specific isozyme(s) is(are) present. Thus the presence of a membrane bound carbonic anhydrase which does not react with antibody to one of the soluble isozymes is evidence of a different isozyme and has been designated CA-IV. This is exemplified by the studies of Wistrand (1979) which showed that certain membranes of human kidney sections were stained by the histochemical method but failed to react with antibodies to CA-I, II or III. The membrane bound form of carbonic anhydrase, unlike the soluble isozymes, is completely resistant to inhibition by halide anions (March, 1980). The amino acid compositions of the CA-IV type isozyme of bovine lung (Whitney and Briggle, 1982), rat saliva (Feldstein and Silverman, 1984), ovine parotid glands (Fernley et al., 1984) and rat brain myelin (Sapirstein and Lees, 1978) as well as that of human kidney (Wistrand, 1984) all differ from their respective soluble isozyme forms. A comparison of the amino acid levels of human CA-IV with those of the soluble isozymes is presented in Table 2. It is apparent that extensive differences exist. The comparisons made Table2. A comparisonof differencesin the aminoacidcompositions of humancarbonicanhydrases° Residue Differencesfrom CA-IV Amino No, in acid CA-IV CA-1 CA-II CA-III Asx 22 +9 +8 +7 Thr 14 +1 -2 -2 Ser 20 +8 -2 -4 Glx 28 -7 -2 -8 Pro 14 +4 +3 + 10 Gly 32 - 16 - 10 - 14 Ala 20 - I -6 -5 Val 14 + 3 - 1 - I Met 3 - 1 - 2 0 lie 11 -4 -4 0 Leu 24 -3 +I 0 Tyr 8 0 0 +1 Pbe 10 0 +2 +2 Tip (7) -- I +1 +1 His 6 +5 +6 +7 Lys 15 +3 +9 +4 Arg 13 -5 -5 +1 Cys 2 - 1 - 1 +3 qt was assumedthat the sum of the molefractionsof aminoacids reported by Wistrand (1984) were equal to 260 amino acid residues. Allcalculatedlevelswereconvertedto integralamounts and it was also assumed that CA-IV contained 7 residues of tryptophan. The estimationsmadelead to somediscrepanciesin the residuenumbersexpected.

104

HAROLD F. DEUTSCH

relate to an enzyme containing about 260 amino acid residues even though the CA-IV forms appear to have higher molecular weights (Wistrand, 1984; Whitney and Briggle, 1982) and may be also associated with considerable amounts of carbohydrate (Whitney and Briggle, 1982). The amino acid compositional data strongly suggest that the membrane forms of the isozyme are products of a genetic locus differing from those coding for CA-I, CA-II and CA-Ill. Definitive proof of this must await sequence analyses of the CA-IV type enzymes. Due to the difficulties in isolating these components in pure form it is most likely that such data will be obtained from their requisite nucleotide sequences. Such studies may perhaps give evidence of more than one additional form of membrane bound isozyme. Clarification of the roles of membranebound carbonic anhydrases are needed to determine how hydrogen and bicarbonate ion and CO2 transfers across membranes are facilitated in seeking explanations for a variety of poorly understood physiologic mechanisms relating to ion transfers. Due to the simultaneous presence of the CA-I and/or CA-II forms of the isozyme in the tissues being investigated for CA-IV, it will be difficult to delineate the specific physiologic role for a given isozyme.

GENETIC VARIATIONS

A relatively large number of amino acid substitutions have been noted both in human and animal carbonic anhydrases. These are summarized in Table 3. It is apparent that the ones found usually involve a substitution which results in a charge modification. This has resulted from the almost exclusive use of the electrophoretic technique along with staining either for hydratase or esterase activity to detect isozymes with different mobilities from normal. An immunological detection method was employed following electrophoretic separation to detect a polymorphism in the erythrocyte carbonic anhydrases of American Blacks that was never observed by the usual staining methods (Moore et al., 1971). In this case the substitution of glutamic acid for lysine at residue 17 of CA-If resulted in a modification of the charge of the protein so that it migrated in the position of CA-I. Since the CA-I and CA-II isozymes have different

immunological properties the electrophoretic area usually staining for and reacting with antibody to CA-I also reacted with antibody to CA-II in this instance. In the case of equine CA-I forms, sequence analysis of the most common low activity isozyme, originally designated CA-D, revealed a series of conservative substitutions (Jabusch et al., 1980). It is most likely that there are a relatively large number of human carbonic anhydrases which possess similar "silent" substitutions. They will not be readily detected since sequence analysis of isozymes from single individuals are not usually performed. In addition, carbonic anhydrases are under the control of codominant, autosomal alleles. A carbonic anhydrase preparation showing a single electrophoretic component could contain two isozymes differing by a "silent" substitution. A carbonic anhydrase isolated from a single animal which showed two low or two high activity isozymes differing in electrophoretic properties could be used to separate the product of a single allele and provide material that was unquestionably pure from a sequence standpoint. In sequencing an isozyme isolated from pooled erythrocytes or other tissues, the presence of a mutant form with a conservative substitution will most likely not be noted if it constitutes less than 10% of the preparation. An exception to this may be the separation by high performance liquid chromatography (HPLC) of peptides from proteolytic digests of such material. Since the syntheses of carbonic anhydrases are under control of codominant autosomal alleles the levels of two different peptides in enzyme from single individuals should be equal. Using the HPLC method, Hewett-Emmett et al. (1983) have discovered a polymorphism of human CA-Ill involving a Leu to lie interchange at residue 31. More widespread use of HPLC methods in resolving peptides appears to present the most likely approach to detect such conservative substitutions. While the mutant and polymorphic forms of carbonic anhydrases are of evolutionary interest no distinct physiological roles for the different isozyme forms of CA-I, CA-II and CA-Ill have as yet emerged. The synthesis of CA-Ill, however, may be linked to that of CA-II in some manner. The muscle isozyme has been found in human erythrocytes at a level of about 0.15 mg per g of hemoglobin. This is approximately 8% of the level of CA-II and 1% of

Table 3. Some naturally ocurring a m i n o acid substitutions noted for carbonic anhydrase isozymes ~ lsozyme H u m a n CA-1

Equine CA-I

Noted a m i n o acid substitutions 8

Asp

--, Gly

76

Arg

100

Thr

--~ Lys

225

Gin

---* Gin Arg ---* Lys

253

Gty

-* Arg

255

Thr

~

54 82 183 224

Ala Gly Ser Ser

~ --~ ~ ~

65 115 212

Gly Ser Cys

~ Ser ---* His --~ T y r

~

237

Pro

~

Asp Cys Arg Ala

Human CA-II

18

Lys

Equine CA-II

2

Ac. Ser

Glu

Bovine C A - I I

56

Arg

~

Gin

Human CA-Ill

31

Val

~

lle

~

Ac. Thr

86

Asp ---* Gly

236

Asp ~

Val

81 157 222

Asp ~ Leu ~ Gln ~

Gly Gly Arg

Arg

His 182 A r g

"The most c o m m o n residue noted is shown to the left of the arrows.

253 ~

Asn ~ Asp Cys

Carbonic anhydrases CA-I (Carter et al., 1984). These workers found that in the case of a family carrying a CA-II deficiency gene there was a doubling in the level of erythrocyte CA-Ill. It appears that a deficiency of erythrocyte CA-II is the primary defect in the autosomal recessive syndrome of osteopetrosis ("marble bone disease") with renal tubular acidosis and cerebral calcification (Sly et al., 1983; Tashian et al., 1984; Sly et al., 1985). Heterozygotes have about half of the normal levels of this isozyme, Conroy and Maren (1985) have presented a method for the detection of individuals homozygous or heterozygous for this condition based upon the differential inactivation of the CA-I and CA-II isozymes by bromopyruvate (Gothe and Nyman, 1972) and by iodide (Maren and Sanyal, 1983). Since heterozygous individuals are relatively asymptomatic an easy method of detection of such individuals is important. Conroy and Maren (1985) have also found that the erythroyctes of homozygous individuals had distinct elevations in their CA-I activities. While the findings of Sly et al. (1985) are of great interest it is difficult to link the renal findings in osteopetrosis with a simple deficiency of the soluble cytoplasmic kidney enzyme analogous to erythrocyte CA-II. A consideration of the possible connection between the renal tubular acidosis and the erythrocyte CA-II deficiency in this disease has been presented by Maren (1985). In pursuing the relationship between osteopetrosis and CA-II deficiency, experimental animal models are of interest. Osteopetrosis is an autosomal recessive disease in several species (Marks and Walker, 1976). In microphthalamic mice it is characterized by defective osteoclasts and a concomitant severely reduced bone resorption. Jilka et al. (1985) in studies of the microphthaimic (mi/mi) mouse detected no apparent abnormality of the carbonic anhydrases in the erythrocytes of these animals and also found that CA-II was present in their osteoclasts. Thus it appears that unlike the human, a CA-II deficiency is not involved in the etiology of the osteopetrosis in the microphthalmic mouse. Studies to date have not indicated any physiological disturbances accompanying a deficiency of erythrocyte CA-I (Kendall and Tashian, 1977). However, Shapira et aL (1984) and Kondo et al. (1978) have reported that in some patients with distal type renal tubular acidosis the CA-I isozyme possessed a lower activity. The latter workers indicated that the CO2 hydration activity of this isozyme could be restored to the normal range by the addition of zinc. While these reports appear to contrast with the work of Kendall and Tashian (1977) and Shepherd and Spencer (1984) it should be remembered that all of these investigations relate to CA-I in erythrocytes and that the effects of CA-II deficiency and the possible involvement of an unusual form of CA-I in individuals with renal tubular deficiency present interesting avenues of exploration of physiological roles of these isozymes. The situation for CA-I is particularly difficult to comprehend because most present evidence indicates that CA-II is the only soluble enzyme present in the kidney (Wistrand, 1980; Dobyan and Bulger, 1982; Spicer et al., 1982).

105

TISSUE LEVELS--FACTORSAFFECTING SYNTHESIS The amounts of the carbonic anhydrase in erythrocytes show marked variations in different species. In humans the level of CA-I is about 12mg per g of hemoglobin which is about 6 times higher than the level of CA-II. Human erythrocytes also possess small amounts of CA-Ill, approximately 0.15 mg per g of hemoglobin (Carter et al., 1984b). The levels of this isozyme in muscle, however, are much higher and may range up to 10% of the total protein in the cytosol. Only trace amounts are found in human tissues other than skeletal muscle (Jeffery et al., 1980). The CA-III isozyme appears to be almost exclusively localized to type I, i.e. red muscle fibers (V~i/in/inen et al., 1982, 1985; Shima et al., 1983). Species such as the horse appear to possess relatively lower levels of erythrocyte CA-I than humans but essentially equal amounts of the CA-II and CA-I isozymes (Deutsch et al., 1972). Some species such as the ox and sheep show only the presence of the high activity form in erythrocytes but relatively high levels of the CA-I type isozyme in the tissues of the rumen (G/ilfi et al., 1982). The reasons for such species variations are not known. The erythrocyte levels of human CA-I appear to show more physiological variations than those of CA-II. The concentrations of erythrocyte CA-I are markedly decreased in thyrotoxicosis and return to normal with treatment (Funakoshi and Deutsch, 1971). This isozyme is also found in relatively low amounts in the erythrocytes of neonates (Funakoshi and Deutsch, 1971; Shepherd and Spencer, 1984). The levels of CA-Ill in the muscles of male and female rats are essentially the same. However, this isozyme was found in high concentration only in the liver cytosol of male rats, accounting for about 8% of the protein (Carter et al, 1984). The liver cytosol of adult female rats possessed about 3% of the levels found in males, i.e. CA-Ill constituted less than 0.3% of their liver cytosol proteins. The administration of testosterone to ovariectomized adult female rats induced a 4 to 5 fold increase in their liver CA-Ill levels, to about those found in the analagous tissue of immature male rats. Castration and treatment of male rats with estradiol was followed by a 75-95% reduction in their liver CA-Ill levels. Although a simple linear dose-dependent relationship between serum testosterone and CA-Ill levels did not appear to exist, the synthesis of this isozyme in liver appears to be under androgenic control. Hypophysectomy had no effect on the concentration of CA-Ill in male rats but in females resulted in the elevation of this isozyme in liver to the levels found in males (Jeffery et al., 1984). These findings present interesting avenues of exploration regarding the control of expression of the gene for CA-Ill by various steroids and of CA-I by thyroid hormones. The CA-Ill isozyme isolated from rat muscle and the inducible liver form appear to be the same protein (Carter et al., 1981). The isolates from both tissues show almost identical patterns of inhibition by anions and by sulfonamides (Sanyal et al., 1982). The erythrocyte form of CA-Ill also appears to be identical with the muscle isolates (Carter et al., 1984).

106

HAROLD F. DEUTSCH ENZYMATIC ACTIVITY

The rates of catalysis of the hydration of CO: and of the dehydration of bicarbonate by the carbonic anhydrase isozymes show a wide range of activities with the CA-II forms showing the highest enzymatic activity known. Since the three well-known isozymes have homologous structures a great deal of interest has centered on attempting to relate the marked variations in activity with specific structural differences. In addition to the reversible catalysis of the hydration of CO2, the carbonic anhydrases also catalyze the hydration of various aldehydes and of pyruvic acid and the hydrolysis of a series of rather divergent compounds (Pocker and Sarkanen, 1978). It has recently been noted that upon the hydrolysis of certain aromatic type esters equine CA-III, but not the CA-I and CA-II isozymes, become acylated (Nishita and Deutsch, 1986). This is a property shared by the homologous protein ubiquitin (Jabusch and Deutsch, 1985). Rabbit CA-III has been also found to exhibit acid phosphatase activity (Pullan and Noltmann, 1985). The analogous equine enzyme possesses both alkaline and acid phosphatase activity (Nishita and Deutsch, 1986). It is interesting that ubiquitin has also been found to show the latter action (Taniguchi and Matsumoto, 1985). There appears to be no physiological counterpart to these activities since only aromatic acyl- and phosphate esters appear to be hydrolyzed. Interest in the elucidation of the catalytic mechanisms for the reactions catalyzing the CO2 conversions have been centered on (1) The addition of H20 to the zinc moiety. (2) The splitting of a hydroxyl bond in the H2Cbenzyme-substrate complex. (3) The transfer of the hydrogen ion released from the active site to specific isozyme residues and then to the solvent medium. (4) the interconversions of CO2 and HCO3. Mechanistic models have been recently elaborated in some detail by Coleman (1984), Lindskog et al. (1984), Pocket and Diets (1984), Koenig and Brown (1984) and Cook and Allen (1974) which also consider the results of other investigators. Included in these considerations is the attachment of a molecule of H20 to the zinc atom which is possibly linked in a hydrogen-bonded network to threonine 199-glutamic acid 106, the splitting of the water, and in CA-I and CA-II, the transfer of the hydrogen ion formed, from the metal to a titrable histidine, with its rapid transfer to components in the solvent. This transfer of the hydrogen ion to a histidine appears to limit the rate of maximal catalysis in the highly active CA-II isozyme. The CA-II isozymes of all species have a single titrable histidine (residue 64) in the active site which appears to effect this hydrogen transfer. Most CA-I isozymes have three such histidines, residues 64, 67 and 200. However, equine CA-I has a glutamine instead of histidine at position 67 (Jabhsch and Deutsch, 1980). This substitution has small but demonstrable effects on the acidic dissociation of the C-2 protons of histidines 64 and 200 of equine CA-I (Forsman et al., 1983). It is possible that these differences in equine CA-I account

for the relatively lower hydration activities of these isozymes as compared with the analogous isozymes of other species (Deutsch et al., 1972). The presence of a single titrable histidine (residue 64) in the active site of CA-II which is considerably more acidic than the same residue in the CA-I type isozymes, along with the additional two histidines (residues 67 and 200), in CA-I may account for the marked differences in the activity of these two forms. However, the pathway for the proton transfer in the carbonic anhydrases remains speculative. Since the H20-splitting step appears to depend in some measure on a titrable histidine close to the zinc, the absence of such a histidine in CA-Ill may indicate that the low activity of this isozyme is due to this structural feature, In this case the hydrogen ion transfer would be inefficiently made directly to a component of the solvent. Following the hydrogen ion transfer, the zinc-bound hydroxyl group reacts with CO2 to form bound bicarbonate. Upon reaction with a molecule of H20 the bicarbonate ion is released. The activities of various carbonic anhydrase isozymes along with data relevant to their inhibition are presented in Table 4. It is apparent that the equine erythroycte forms are considerably less active than their own mammalian counterparts. A rather wide range of activities for the CA-Ill forms is also noted, the chicken isolate appearing to possess from 25 to 50 times the activity of the least active mammalian isozymes. ENZYME INHIBITORS

Studies of the effects of anions, sulfonamides and of acylation on the enzymatic activities of the various isozymes are directed to providing information regarding the mechanism of their action and of relating the influence of specific residues to these activities. The effects of amino acid substitutions and derivatizations of the side chains of amino acids comprising active-site residues is of particular interest. Some of the parameters relating to the inhibition of the three common isozymes are included irl the data of Table 4. The inhibition by anions depends on the displacement of the hydroxyl group bound to the zinc (Lindskog, 1982). Sulfonamides such as acetazolamide are strong inhibitors and the latter compound is bonded to the zinc and to the active-site exposed nonpolar side chains of residues 91, 121 and 131 in the case of CA-I and CA-II (Kannan, 1980). The low activity CA-I isozymes are less sensitive to inhibition by acetazolamide than the CA-II forms. An exception to this is equine CA-I. Chegwidden et al. (1986) have suggested that the similar inhibitions noted for equine CA-I and CA-II may relate to these isozymes having identical sulfonamide binding residues whereas in the analogous human forms they are different. From the data of Table 4 it is apparent that CA-III is only weakly inhibited by acetazolamide. This may reflect the presence of arginine at residue 91 instead of a hydrophobic amino acid in mammalian CA-I and CA-II isozymes. Equine CA-III is particularly resistant to the action of this sulfonamide as compared to the analogous bovine and chicken isozymes. This may relate to the presence of tyrosine at residue

Carbonic anhydrases

107

Table 4. Variationsin the activitiesand inhibitionsof carbonicanhydraseisozymes Ki (uM) Turnover lsozyme (sec-I × 10 3) Acetazolamide Cyanate Chloride CA-I Human 29 0.2 0.7 6.0 Lapine I0 0.2 Equine~ 2.4 0.02 CA-II Human 236 0.01 20 200,000 Lapine 140 0.02 Equine 42 0.02 Bovine 93 94,000 Galline 0.03 9 CA-Ill Human~ I 50 Equineu 7.8 1800 Bovine 0.5 Galline~ 27 7 Rattine 100 6.70 11,000 Feline 1.0 306 0.52 5,900 The data obtainedat 0-2'~Care taken for bicarbonatedehydrationby Chegwidden et al. (1986)and for CO2hydrationby Sanyalet al. (1982)and Marenand Sanyal (1983). ~Bicarbonatedehydration. 131 in the equine enzyme (Chegwidden et al., 1986). The CA-Ill like the CA-I isozyme is more strongly inhibited by cyanate and chloride than CA-II. Maren et al. (1976) have indicated that human CA-I is inhibited by the levels of chloride and bicarbonate occurring in cells to such an extent that its biological role is not likely that of a hydratase. However, in CA-II deficiency in humans there appears to be no abnormality in the CO2 transport by erythrocytes. This suggests that CA-I can function adequately in such a role (Tashian, 1984). Little data exists on the kinetic properties of membrane forms of carbonic anhydrase. The bovine lung isolate was found to be inhibited by acetazolamide nearly as well as the CA-II erythrocyte form (Whitney and Briggle, 1982). The use of detergents in the isolation of the membrane isozymes makes difficult the interpretation of experimental data relating to their kinetic parameters. SELECTED PHYSIOLOGIC ROLES A variety of studies are being directed to determine if carbonic anhydrases function in metabolic roles other than the classic reversible catalysis of CO2 hydration, bicarbonate dehydration and likely roles in facilitated diffusion of CO2 and of hydrogen ion production. These type investigations usually determine whether a sulfonamide has an effect on a particular biochemical reaction. Thus Coulson and Herbert (1984) have shown that pretreatment of lizards with a carbonic anhydrase inhibitor prior to the administration of pyruvate resulted in a marked increase in alanine synthesis as compared with an uninhibited control. If one assumes that the inhibitor did not directly affect pyruvate carboxylase and thus prevent the formation of oxalacetate, it appears that the bicarbonate level of the cells was lowered to such an extent that little oxalacetate was formed and pyruvate was shunted largely into the transamination reaction to form alanine. Gluconeogenesis from lactate also involves the carboxylation of pyruvate. In

the presence of a carbonic anhydrase inhibitor, alligators showed a diminished capacity to resynthesize glucose from lactate (Herbert et al., 1983). Bicarbonate is also required in fatty acid synthesis in the carboxylation of acetate to form malonate. Carbonic anhydrase inhibitors have been shown to block fatty acid synthesis, presumably by the lowering of intracellular bicarbonate levels (Woodbury, 1980; Herbert and Coulson, 1984). The rate of citrulline synthesis by guinea pig liver mitochondria is also strongly inhibited by azetazolamide, due apparently to the lowering of bicarbonate levels and interference with the formation of carbamoyi phosphate (Dodgson et al., 1984). These workers noted that urea formation by intact hepatocytes could be partially inhibited by ethoxzolamide but not by acetazolamide. All of these metabolic effects appear to result from the lowering of the concentration of intracellular bicarbonate. It does not appear that the usual therapeutic uses of carbonic anhydrase inhibitors in various clinical disorders are accompanied by any significant metabolic disturbances in the areas just discussed.

SULFHYDRYL CHEMISTRY

Recent studies of the sulfhydryl chemistry of the carbonic anhydrases are providing insights on possible functions of their cysteine residues. The erythrocyte isozymes, CA-I and CA-II, usually possess a single such residue although bovine CA-II has none (Nyman and Lindskog, 1964; Sciaky, 1976). A minor form of equine erythrocyte CA-I (Jabusch et al., 1980) and one of equine CA-II (Deutsch et al., 1977) have two cysteines. In the case of the latter isozyme, the cysteine, which has replaced the usual arginine at residue 180, can form a mixed disulfide with glutathione. This cysteine should be the N-terminal residue of the short F-helix and is on the surface of the protein far from the active site (Nostrand et al., 1974). The complex with glutathione results in a

108

HAROLD F, DEUTSCH

lowering of the isoelectric point of this isozyme from ing derivatization of its two reactive cysteines with 9.00 to 8.52. No changes in hydration activity or in iodoacetamide (Nishita and Deutsch, 1981). immunologic properties appear to result. Engberg and Lindskog (1986) have recently found The high activity erythrocyte isozymes of the frog that reaction of the two readily accessible sulfhydryls (Bundy and Cheng, 1976), turtle (Hall and Shraer, of bovine CA-III with 5,5'-dithiobis(2-nitrobenzoate) 1979) and chicken (Bernstein and Shraer, 1972) all also has no effect on its hydration activity. However, have multiple cysteine residues as compared to a treatment with a large excess of this reagent results in single such residue in some mammalian CA-II iso- • a further slow derivatization of 2 or 3 of the remainzymes. ing thiols that is accompanied by an almost 2-fold Kondo et al. (1974) have described a form of increase in hydratase activity. A 120% activation was erythrocyte CA-I in patients with primary al- noted with 2,2'-dithiopyridine and 400% with dosteronism in which the single cysteine residue methyl-methanethiosulfonate. No modification of acappears to be disulfide-bonded with glutathione. Al- tivity resulted from prolonged treatment with iothough the erythrocyte level of this isozyme is in doacetate, iodoacetamide or N-ethyl-maleimide. the normal range in these patients, it possesses about Elucidation of the structural changes in CA-III half of the normal esterase activity. Upon reduction that accompany derivatizations which lead to inwith dithiothreitol the enzymatic acitivity returns to creases or decreases in activity will most likely require normal. study of crystalline derivatives. This would possibly The glutathione containing isozyme shows a de- permit elucidation of how specific steps in the reaccreased affinity for acetazolamide. Surprisingly the tion mechanisms of the enzyme are affected by expected lowering of the isoelectric point of this form specific modifications. of the enzyme was not observed. A peptide map Muscle carbonic anhydrase appears to show some of the tryptic digest showed two peptides in the similarities to the enzyme isolated from parsley. The glutathione containing CA-I not present in the under- latter isozyme contains 7 cysteines, has nO esterase ivatized, i.e. normal, isozyme. This finding is also of activity and its hydratase activity is poorly inhibited interest since the portion of human CA-I which by acetazolamide (Tobin, 1970). The spinach enzyme contains the single cysteine residue appears in the appears to have similar properties (Pocker and Ng, so-called insoluble "core" peptides of the tryptic 1974; Kandel et al., 1978). Other carbonic anhydrases digest (Lin and Deutsch, 1973). It was earlier shown isolated from peas (Kisiel and Graf, 1972) and lettuce by Rickli and Edsall (1962) that the cysteine of (Walk and Metzner, 1975) are also characterized by human CA-I was not accessible to the usual al- relatively higher amounts of cysteine than the erythkylation with iodoacetate. Later delineation of its rocyte forms. The isozyme isolated from hagfish crystal structure (Notstrand et al., 1974) showed that erythrocytes appears to contain about 260 amino acid cysteine 212 is part of strand 7, a large fl-structure, residues and to possess 4 cysteines (Carlsson et al., and that it lies within the center of the molecule and 1980). Although the cysteine level is more similar to thus most likely would not be readily available to that of CA-III its content of other amino acids, form a disulfide with glutathione. Determination of particularly the level of serine, as well as its hydration the composition and sequence of the variant tryptic activity, indicates that it is more closely related to the peptides of the CA-I from patients with primary mammalian CA-I type isozymes. However, unlike the aldosteronism noted by Kondo et al. (1984) would latter enzyme it is not readily inhibited by anions. resolve the question of whether this isozyme might be Carlsson et al. (1980) suggest that since it is the only a polymorphic form of CA-I. enzyme of erythrocytes it should be a form that The sulfhydryl chemistry of CA-III appears to be unlike CA-I is not readily susceptible to inhibition by more complex than that of the erythrocyte forms. chlorides. Both the equine (Wendorff et al., 1985) and the The bull and the tiger shark erythrocyte carbonic bovine (Engberg et al., 1985) isozymes contain 5 anhydrases are distinguished from vertebrate forms cysteine residues. The CA-III of other species also of the enzyme not only by possessing molecular possesses multiple cysteine residues, the pig 5 (Pullan weights 6000-9000 higher but also having 25 and 18 and Noltmann, 1984), rabbit 6 (Register et al., 1978), cysteine residues, respectively, all of which exist in cat 4 (Sanyal et al., 1982) and human 4 (Carter et al., disulfide form (Maynard and Coleman, 1971). The 1979). Eventual sequence analyses may well disclose carbonic anhydrase of teleosts resembles the mamthat all species contain 5 such residues. The 5 cys- malian CA-I isozyme in its hydration activity and teines in equine (Wendorff et al., 1985) and in bovine also possesses only 1 cysteine residue which is avail(Engberg et al., 1985) CA-III are localized in identical able for derivatization after denaturation (Sanyal et positions, i.e. residues 66, 183, 188, 203 and 206. Two al., 1982a). Clarification of the evolutionary aspects of these, residues 183 and 188, react readily with of the carbonic anhydrases will require some sealkylating agents, whereas derivatization of the quence analyses of the enzymes isolated from lower others proceeds very slowly or requires denaturing forms. conditions. Alkylation of the two reactive cysteines in the equine protein prevents the dimerization noted by CHEMICAL AND ENZYMATIC MODIFICATIONS other workers (Koester et al., 1978; Carter et al., OF CA-Ill 1979; Register et al., 1978) but has no effect upon its Treatment of human and bovine CA-III with hydration activity (Nishita and Deutsch, 1981). The dimerized form of cat CA-III, however, shows a 2,3-butanedione has been reported to result in a significant decrease in activity (Sanyal et al., 1982). pronounced activation of both its bicarbonate dehyThe equine CA-III can be readily crystallized follow- dration and esterase activities (Tashian et al., 1984;

Carbonic anhydrases Chegwidden et al., 1984). In the case of the analogous chicken isozyme only the esterase activity was affected (Chegwidden et al., 1986). No effect of 2,3-butanedione on human CA-I and CA-II was noted. It was suggested that the activation of the muscle isozyme could be associated with modification of arginine residues 67 and 91 which are presumed to be in the active-site area of CA-Ill. Only the latter type isozymes possess arginine residues at these positions. Furthermore, the modification with butanedione reduced the Is0 for acetazolamide to about 20% of the level of the native enzyme in the case of human and chicken CA-Ill. This would suggest a strong role for residue 91, one of the three found to be involved in the binding of azetazolamide by carbonic anhydrase (Kannan et al., 1977). Contrasting with the above result is the report of Pullan and Noltmann (1984) that the CO2 hydratase and esterase activities of porcine CA-Ill were not affected by relatively low levels of phenylglyoxal whereas its acid phosphatase was strongly inhibited. The use of phenylglyoxal levels 50-100 times those employed to inactivate the phosphatase activity, i.e. 1.2 mM, led to losses of both esterase and hydratase activities. The difference in these results may be due to the use of 2,3-butanedione, a reversible modifier of arginine residues, in one case and of phenylglyoxal in the other. However, when 2,3-butanedione was used by Engberg and Lindskog (1986) under conditions similar to those employed by Tashian et al. (1984) and Chegwidden et al. (1986), some inhibition of the hydratase activity of bovine CA-Ill was noted. It appears that more detailed kinetic studies that correlate with precise determination of the modifications induced by guanidinium complexing agents will be required to clarify the role of specific arginine residues in the activity of CA-Ill type isozymes. It has also been found that the dehydrations of bicarbonate catalyzed by CA-I, CA-II and CA-Ill are all strongly inhibited by carbamoyl phosphate (Carter et al., 1984). It is unusual that an inhibitor would show similar characteristics for all three isozymes. While this report is of great interest the result appears capable of various interpretation. Carbamoyl phosphate at neutral and alkaline pH readily forms cyanate (Jones and Lipmann, 1960; Halmann et al., 1962; Allen and Jones, 1964). The half-life of carbamoyl phosphate at neutral pH is about 40 rain. Since milli-molar levels of carbamoyl phosphate were employed by Carter et al. (1984) and the K,. (#M) constants for the three carbonic anhydrases range from 0.5 to 20, it would appear that sufficient N C O would have been generated to strongly inhibit all three of the isozymes. Pertinent to these considerations is the covalent modification of equine CA-Ill resulting from its interaction with carbamoyl phosphate (Nishita and Deutsch, 1986). When this enzyme is incubated with carbamoyl phosphate at essentially the same concentration as employed by Carter et al. (1984) a series of more anodic components are formed. These reflect the carbamoylation of the E-amino group of lysine. When an average of 2 lysines are carbamoylated there is a loss of about 60% of the hydration activity. Further carbamoylation, to an average of 6 lysyl groups, results in an additional 10% loss of en-

109

zymatic activity. These losses are considerably less than those experienced by Carter et al. (1984). In the experiments of Nishita and Deutsch (1986) the carbamoylated species of CA-Ill isozyme were separated by isoelectric focusing. This would result in the removal of dissociable cyanate formed by the breakdown of carbamoyl phosphate and the inhibition noted would only be due to the effect of the carbamoylation of lysine residues. The results of Carter et al. (1984) could be expected to be due both to anion inhibition and carbamoylation. It is interesting that the carbamoylation of several lysines in equine CA-Ill results in a loss of over 60% of its activity. The amidination of all of the lysine groups in human CA-I resulted in no loss of either its esterase or hydration activity (Whitney et al., 1967). A consideration of the amino acid residues most directly associated with the active centers of the different carbonic anhydrases does not suggest reasons for the sensitivity of equine CA-Ill to carbamoylation of some of its E-amino lysyl residues. Initial attempts to determine which lysine residues in equine CA-Ill had been derivatized were not successful (Nishita and Deutsch, 1986). The cleavage of various p-nitrophenyl esters by equine CA-III is also accompanied by the formation of a series of more anodic components (Nishita and Deutsch, 1986). The result for an experiment using p-nitrophenyl butyrate is shown in Fig. 1. The result appears to be due, as in the case of ubiquitin (Jabusch and Deutsch, 1985), to the acylation of E-amino lysyl residues. Although the equine CA-Ill possessed both alkaline and acid phosphatase activities for p nitrophenyl phosphate (Nishita and Deutsch, 1986), no charge modification of the protein was effected by this substrate. Direct sequencing of the acylated equine CA-III could not be carried out as in the case of ubiquitin (Jabusch and Deutsch, 1985) because of the blocked N-terminus. Peptide maps of the individual components of the modified CA-III indicated that the acylation was variable, i.e. although an average of 2 lysines of the M~ component were acylated, more than 2 individual lysines had reacted (Nishita and Deutsch, 1986). Extensive sequence analyses will be required to delineate which lysine residues accept the acyl groups ofp-nitrophenyl esters and the extent to which specific residues are acylated.

Froe,,o M, Isoel. pl-~ 7.~0 1.5

E t-

°/°Total: eAc-Lys:

30 2

M2

6.50 3/, 3

5i 5 22

1.0

O

<

0.5

Elution volume Fig. 1

5

/, 6

I10

HAROLD F. DEUTSCH SUMMARY

Some o f the current studies of carbonic anhydrases are directed to the genetic mechanisms underlying their synthesis. D e t e r m i n a t i o n o f the structure o f their genes will p r o b a b l y most readily resolve the question of whether the m e m b r a n e b o u n d forms o f the enzyme represent products of additional loci o t h e r t h a n those of the three well-known soluble forms. Extensions of o u r knowledge of the sequences of these isozymes as well as those from lower animals a n d from plants will m a k e possible a more precise evaluation of the extent of the multigene aspects of these proteins a n d their evolutionary backgrounds. Studies o f the interrelationships o f the regulation of the transcriptional a n d translational processes of the well-known isozymes and in particular the effects of h o r m o n e s will be o f interest. Insights into modifications of the isozymes' synthetic processes occurring in various diseases should also be forthcoming from these studies. In addition to the above the applications of what are perhaps today s o m e w h a t classical m e t h o d s of protein chemistry will be needed to explore the reasons for the changes in activity a c c o m p a n y i n g the sequence variations of the different isozymes, the decreases or increases in activity a c c o m p a n y i n g derivatizations of specific residues and the reasons for the differences in the activity of different inhibitors o n the various isozymes. The b r o a d specificity o f these enzymes for different substrates and the ability of C A - I l l to hydrolyze various phenyl esters a n d in some cases to become derivatized also present problems in protein structural chemistry. In terms of the latter reactions, the m e a n i n g o f the relationships of these activities to those o f the protein ubiquitin, which is h o m o l o g o u s to C A - I l l , needs clarification. It would a p p e a r that various of the protein structural studies will be aided by crystallographic investigations o f not only C A - I l l but of various of its derivatives which undergo either increases or decreases in activity. The above areas of studies present a wide variety o f problems for workers in various disciplines a n d b a c k g r o u n d s w h o are interested in the carbonic anhydrases. Acknowledgements--The author wishes to thank the Alex-

ander von Humboldt Foundation and the University of Wisconsin Graduate School for financial support during the period in which this review was prepared. Grateful acknowledgement is also extended to Professor Peter Traub for placing the facilities of his institute at the author's disposal and for his critical comments on this manuscript. REFERENCES

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Carbonic anhydrases Purification and properties of a polymorphic high activity equine erythrocyte carbonic anhydrase. J. biol. Chem. 252, 555-559. Deutsch H. F. (1984) Primary structures and genetic changes in mammalian carbonic anhydrases. Ann. N.Y. Acad. Sci. 429, 183-194. Dobyan D. C. and Bulger R. E. (1982) Renal carbonic anhydrase. Am. J. Physiol. 243, F311-F324. Dodgson S. J., Forster R. E. II and Storey B. T. (1984) The role of carbonic anhydrase in hepatocyte metabolism. Ann. N.Y. Acad. Sci. 429, 516-524. Dworkin-Rastl E., Shrutkowski A. and Dworkin M. B. (1984) Multiple ubiquitin mRNAs during Xenopus laevis development contain tandem repeats of the 76 amino acid coding sequence. Cell 39, 321-325. Engberg P., Millquist E., Pohl G. and Lindskog S. (1985) Purification and some properties of carbonic anhydrase from bovine skeletal muscle. Archs Biochem. Biophys. 241, 628~38. Engberg P. and Lindskog S. (1986) Activation of bovine muscle carbonic anhydrase by modification of thiol groups. Eur. J. Biochem. 156, 407-412. Feldstein J. B. and Silverman D. N. (1984a) Properties of carbonic anhydrase from the saliva of the rat. Ann. N.Y. Acad. Sci. 429, 214-215. Feldstein J. B. and Silverman D. N. (1984b) Purification and characterization of carbonic anhydrase from the saliva of the rat. J. biol. Chem. 259, 5447-5453. Fernley R. T., Congiu M., Wright R. D. and Coglan J. P. (1984) Characterization of a high molecular weight carbonic anhydrase from ovine parotid glands. Ann. N.Y. Acad. Sci. 429, 212-213. Ferrell R. E., Stroup S. K., Tanis R. J. and Tashian R. E. (1978) Amino acid sequence of rabbit carbonic anhydrase II. Biochim. biophys. Acta 533, 1-11. Funakoshi S. and Deutsch H. F. (1971) Human carbonic anhydrase V. The levels of erythrocyte carbonic anhydrases in various physiologic states. J. Lab. clin. Med. 77, 39-45. Gfilfi P., Kutas F. and Neogrfidy S. (1982) Immunohistochemical detection of bovine ruminal carbonic anhydrase isozyme. Histochemistry 74, 577-584. Giraud N., Marriq C. and Laurent-Tabusse G. (1974) Structure primaire de l'anhydrase carbonic erythrocytaire B humaine. III. Sequences des fragments I CNBr et III CNBr (residues 149 260). Biochimie 56, 1031-1043. Goriki K., Hazama R. and Yamakido M. (1984) Human erythrocyte carbonic anhydrase deficiency in Japanese populations (Hiroshima, Nagasaki). Ann. N. Y. Acad. Sci. 429, 276. Gothe P. O. and Nyman P. O. (1972) Inactivation of human erythrocyte carbonic anhydrase by bromopyruvate. FEBS Lett. 21, 159-163. Graham D., Reed M. L., Patterson B. D. and Hockley D. G. (1984) Chemical properties, distribution, and physiology of plant and animal carbonic anhydrases. Ann. N.Y. Acad. Sci. 429, 222-237. Hall G. E. and Schraer R. (1979) Purification and partial characterization of high- and low-activity carbonic anhydrase isozymes from Malaclemys terrapin centrata. Comp. Biochem. Physiol. 63B, 561-567. Halmann M., Lapidot A. and Samuel D. (1962) Kinetic and tracer studies of the reactions of carbamoyl phosphate in aqueous solution. J. Am. Chem. Soc. 1944-t957. Hansson H. P. J. (1967) Histochemical demonstration of carbonic anhydrase activity. Histochemie ll, 112-128. Heath R., Carter N. D., Jeffery S., Edwards R. J., Watts D. C. and Watts R. L. (1985) Evaluation of carrier detection of Duchenne muscular dystrophy using carbonic anhydrase lII and creatine kinase. Am. J. reed. Genet. 21, 291 296. Henderson L. E., Henriksson D. and Nyman P. O. (1973) Amino acid sequence of human erythrocyte carbonic

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anhydrase C. Biochem. biophys. Res. Commun. 52, 1388-1394. Henriksson D., Tamis R. J. and Tashian R. E. (1980) The amino acid sequence of carbonic anhydrase I from the rhesus macaque. Biochem. biophys. Res. Commun. 96, 135 142. Herbert J. D., Coulson R. A. and Hernandez T. (1983) Inhibition of pyruvate carboxylation in alligators (Alligator mississippiensis) and chameleons (Anolis carolinensis) by carbonic anhydrase inhibitors. Comp. Biochem. Physiol. 75A, 185-192. Herbert J. D. and Coulson R. A. (1984) A role for carbonic anhydrase in de novo fatty acid synthesis in liver. Ann. N.Y. Acad. Sci. 429, 525-527. Hewett-Emmett D., Welty R. J. and Tashian R. E. (1983) A widespread silent polymorphism of human carbonic anhydrase III (31 Ile--Val): Implications for evolutionary genetics. Genetics 105, 409-420. Hewett-Emmett D., Hopkins P. J., Tashian R. E. and Czelusniak J. (1984) Origins and molecular evolution of the carbonic anhydrase isozymes. Ann. N.Y. Acad. Sci. 429, 338-358, Holmes R. S. (1977) Purification, molecular properties, and ontogeny of carbonic anhydrase isozymes: Evidence for A, B and C isozymes in avian and mammalian tissues. Eur. J. Biochem. 78, 511-520. Jabusch J. R., Bray R. P. and Deutsch H. F. (1980) Sequence of the low activity equine erythrocyte carbonic anhydrase and delineation of the amino acid substitutions in various polymorphic forms. J. biol. Chem. 255, 9196-9204. Jabusch J. R. and Deutsch H. F. (1984) Sequence of the high-activity equine erythroycte carbonic anhydrase: N-terminal polymorphism (acetyl-Ser/acetyl-Thr) and homologies to similar mammalian isozymes. Biochem. Genet. 22, 357-367. Jabusch J. R. and Deutsch H. F. (1985) Localization of the lysines acetylated in ubiquitin reacted with p-nitrophenyl acetate. Archs Biochem. Biophys. 238, 170-177. Jeffery S., Carter N. D. and Shiels A. (1984a) Novel purification of carbonic anhydrase 1II from human rat and baboon muscle. Comp. Biochem. Physiol. 78B, 433-436. Jeffery S., Carter N. D. and Wilson C. A. (1984b) Hypophysectomy abolishes sexual dimorphism of liver carbonic anhydrase III. FEBS Lett. 175, 129-130. Jones M. E. and Lipmann F. (1960) Chemical and enzymatic synthesis of carbamoyl phosphate. Proc. natn. Acad. Sci. U.S.A. 46, 1194-1205. Jilka R. L., Rogers J. I., Khalifah R. G. and V/i/in/inen H. K. (1985) Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice. Bone 6, 445-450. Kandel M., Gornall A. G., Cybulsky D. L. and Kandel S. I. (1978) Carbonic anhydrase from spinach leaves. Isolation and some chemical properties. J. biol. Chem. 253, 679-685. Kannan K. K., Notstrand B., Fridburg K., L6vgren S., Ohlsson A. and Petef M. (1975) Crystal structure of human erythrocyte carbonic anhydrase B. Threedimensional structure at a nominal 2.2/~ resolution. Proc. nam. Acad. Sci. U.S.A. 72, 51-55. Kannan K. K., Vaara I., Notstrand B., LSvgren S., Borell A., Fridborg K. and Petef M. (1977) Structure and function of carbonic anhydrase: Comparative studies of sulphonamide binding to human erythrocyte carbonic anhydrases B and C. In Drug Action at the Molecular Level(Edited by Roberts G. C. K.), pp. 73-91. Univ. Park Press, Baltimore. Kannan K. K. (1980) Crystal structure of carbonic anhydrase. In Biophysics and Physiology o f Carbon Dioxide (Edited by Bauer G., Gross G. and Bartels H.), pp. 184-205. Springer, Berlin.

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