Comp. Biochent Physiol., 1977, Vol. 57B, pp. 1 to 7. Pergamon Press. Printed in Great Britain

REVIEW CARBONIC ANHYDRASE HALLIE F. BUNDY Department of Chemistry, Mount St. Mary's College, Los Angeles, CA 90049, U.S.A. (Received 28 September 1976) Abstract--1. Some facets of the comparative biochemistry of vertebrate, invertebrate, and plant car-

bonic anhydrases are reviewed. 2. While all carbonic anhydrases examined are zinc metalloproteins, three prominent molecular changes appear to have occurred during evolution from invertebrates to mammalian vertebrates: loss of the tendency to form high molecular weight aggregates, shortening of the polypeptide chain, and a marked reduction in cysteine content. 3. Attention is given to recent evidence suggesting that carbonic anhydrase plays a role in bone resorption; that enzyme activity is regulated by cyclic AMP-dependent protein kinase; and that there is a functional difference between cytoplasmic and membrane bound carbonic anhydrase. 4. Among plants there appears to be a marked difference in molecular size between monocotyledon and dicotyledon carbonic anhydrase, and some evidence for isozymic forms which may be related to cytoplasmic and chloroplastic carbonic anhydrase.

the carbonic anhydrases from vertebrates, invertebrates, and plants, and with occasional reference to suggested physiological significance. We have not included bacterial carbonic anhydrase. For some recent findings in this area the reader is referred to the reports of Adler et al. (1972), and Brundell et al. (1972).

INTRODUCTION

Carbonic anhydrase (EC4.2.1.1, CO2 + H 2 0 ~ - H + + HCO~) is among the most widely occurring enzymes, having been found in bacteria, lower and higher plants, invertebrates, and vertebrates. The primary physiological function of this ubiquitous enzyme, while still unclear in its details, is thought to reside in the catalysis of the reversible hydration of CO2. Thus it is a potential regulator, depending on its immediate microenvironment, of available concentrations of CO2, H +, HCO3, and CO 2-, and consequently of such processes as gas exchange, acid-base balance, secretion, calcification, and photosynthesis. It is beyond the scope of this brief review to deal with all facets of the chemistry and physiology of carbonic anhydrase. As far as possible key papers have been cited and should be consulted for references and illustrations. Over the years, the enzyme has been the subject of several major reviews. One of the most comprehensive reviews is that by Maren (1967) which includes extensive discussion of the distribution and physiological significance of carbonic anhydrase in plants and animals in general, and in vertebrates in particular. The physical, chemical, and catalytic properties of carbonic anhydrase isozymes have been reviewed by Lindskog et al. (1971), and Carter (1972). The latter emphasizes distribution and physiological significance. The review by Lindskog et al. (1971) includes a discussion of kinetic studies which have contributed to current models of the mechanism of action of carbonic anhydrase. For recent developments in this area see the reports of Lindskog & Coleman (1973), Khalifah (1973), Steiner et al. (1975, 1976), and Tu & Silverman (1975). One might include with these the report of an active site titration for human carbonic anhydrase B (Mendez & Kaiser, 1975). We are concerned here mainly with developments since 1971, with emphasis on the comparative biochemistry of C.P.B. 57/1 ~ A

VERTEBRATE CARBONIC ANHYDRASE

The erythrocyte enzyme

Mammalian erythrocytes provide one of the richest sources of carbonic anhydrase and it is not surprising that it is the carbonic anhydrases from this source which have'been most highly purified,,and thoroughly Characterized. Work during the last two decades on carbonic anhydrases from erythrocytes of man, rhesus monkey, horse, cattle, pig, guinea pig (see reviews by Lindskog et al., 1971, and Carter, 1972) and more recently from sheep (Tanis & Tashian, 1971; Tanis et al., 1974) and European moose (Carlsson et al., 1973) allow some general statements to be made about the structure and enzymatic activity of mammalian erythrocyte carbonic anhydrases. All are monomeric proteins having a molecular weight of about 30,000 with 1 zinc ion per enzymatically active molecule. They are characterized by a low (0-2 residues/molecule) cysteine content. All are powerfully inhibited by acetazoleamide (K i = 10-6-10-SM) and to a lesser extent by cyanide, azide, and halide ions. All are catalysts of the hydrolysis of certain esters, an activity which allows precise spectrophotometric determinations and has proven useful in kinetic studies. On the basis of specific activity in the CO2 hydration reaction, mammalian erythroeyte carbonic anhydrases can be divided into two classes: high activity and low activity carbonic anhydrases. According to the most commonly used system of nomencla1

2

HALLIEF. BUNDY

ture, the high activity form is designated as C, and the low activity form as B. One or both of these two major isozymes may be present in various modified forms accounting for additional polymorphism. Analysis of amino acid composition has revealed a consistent difference between the mammalian high activity and low activity carbonic anhydrases: in all species studied the low activity enzyme has a considerable higher serine content (28-33 residues/molecule) than the high activity enzyme (16-22 residues/molecule). Investigations aimed at establishing amino acid sequence have concentrated on the human and bovine erythrocyte enzymes. Since the review by Lindskog et al. (1971), the complete primary structure for both low and high activity carbonic anhydrase from human erythrocytes has been reported (Anderson et al., 1972; Henderson et al., 1973; Lin & Deutsch, 1973, 1974). While there are a few as yet unresolved differences in the reported primary structures, it appears that there is about 60~o identical homology in the amino acid sequence of the two isozymes. Besides allowing interpretation of the three dimensional structure from X-ray crystallography (Liljas et al., 1972; Notstrand et al., 1975), the primary structures will serve as standards for comparison in studying structural relationships between carbonic anhydrases from a wide variety of species as these become available in highly purified form. Tashian et al. (1975) have begun such a comparison in their study of the evolution of carbonic anhydrase isozymes. The low activity isozyme is apparently absent from the erythrocytes of some mammalian species (see review by Carter, 1972). In addition to cattle, sheep (Tanis & Tashian, 1971), goat (Ashworth et al., 1971), and moose (Carlsson et al., 1973) erythrocytes have been found to contain only high activity forms of carbonic anhydrase. These findings suggest that the absence of the low activity isozyme in erythrocytes may be a characteristic feature of all ruminant species. The absence of a particular isozyme from a given tissue is not in itself evidence for lack of the corresponding gene. While bovine erythrocytes lack low activity carbonic anhydrase, Carter (1971) demonstrated that bovine rumen epithelial tissue contains high levels of a low activity isozyme. Thus it would seem that in this and presumably other ruminant species the lack of erythrocyte low ~/ctivity carbonic anhydrase is due to gene repression. Tanis & Tashian (1971) have suggested that such a gene repression in red blood cells may have occurred early in the divergence of the Artiodactyla. The enzymes from erythrocytes of two species of elasmobranch, the bull shark (Carcharhinus leucas), and the tiger shark (Galeocerdo cuvieri) are the first highly purified carbonic anhydrases obtained from a nonmammalian vertebrate (Maynard & Coleman, 1971). Only one form of the enzyme was found in erythrocytes from both species. The molecular weight, about 36,000 for the bull shark and 40,000 for the tiger shark enzyme is significantly higher than that of the mammalian enzymes. With respect to catalytic properties the enzymes resemble high activity mammalian carbonic anhydrase. However, the serine content is in the range characteristic of the mammalian low activity isozyme. The most striking difference between shark and mammalian carbonic anhydrases

is the high cysteine content of the former. The 18-25 half-cystine residues of shark carbonic anhydrase are apparently cross-linked through intra-molecular disulfide bonds, since neither the native nor denatured enzymes react with sulfhydril reagents. Studies employing cobalt (II) as an active site probe indicate an almost identical coordination geometry at the active site for the shark and mammalian enzymes. It is suggested that, while certain features such as length of the peptide chain and the presence of intramolecular disulfide bonds have undergone considerable change during the evolution of carbonic anhydrase, a specific coordination geometry at the active site has remained a constant feature of the enzymes over the last 400 million years (Maynard & Coleman, 1971). More recent contributions to the study of the comparative enzymology of animal carbonic anhydrases include reports of purification and partial characterization of the enzyme from erythrocytes of the chicken (Gallus domesticus) (Bernstein & Schraer, 1972), turkey (Meleagris gallopavo) (Lemke & Graf, 1974), and the frog (Rana catesbeiana) (Bundy & Cheng, 1976). Again, in each of these nonmammalian vertebrates, only one form of erythrocyte enzyme was found, having catalytic properties resembling those of the high activity mammalian isozyme. The molecular weight of the amphibian and avian enzymes is, like the mammalian enzymes, about 30,000. Thus, it would appear that shortening of the polypeptide chain may have occurred before the divergence of amphibians. That a higher cysteine content is a consistent feature of the carbonic anhydrase of nonmammalian vertebrates is borne out by the data for the amphibian and avian enzymes. The chicken enzyme contains 7 half-cystine residues per molecule. It is as yet not known how many, if any, of these are cross-linked by disulfide bonds in the native enzyme. The amino acid composition of amphibian carbonic anhydrase has not been determined. However, the enzyme has been found to contain 4pCMB-reactive-SH groups per molecule. In contrast to the elasmobranch enzyme, in which all of the cysteine residues appear to be intramolecularly cross-linked as disulfides, the amphibian and avian enzymes lose activity rapidly unless maintained in the presence of a sulfhydril reducing agent, and are inhibited by pCMB. Inhibition by sulfhydril-reactive agents has not been reported for other animal carbonic anhydrases. It appears that cysteine content (both free and in disulfide linkage) of carbonic anhydrase has undergone considerable change during evolution from elasmobranchs, through amphibians and avians, to mammals. The direction of this change is from a large number of intra-molecularly cross-linked cysteines, through a reduction in cysteine content and the appearance o f - S H groups essential for enzyme activity, to the near elimination of cysteine residues (see Lemke & Graf, 1974). The detailed history of this change will require the characterization and sequencing of the enzyme from a wide variety of species. An obvious step in this direction is the isolation and characterization of reptilian carbonic anhydrase. It is of interest that in each of the nonmammalian vertebrates examined, the erythrocytes contain only one type of carbonic anhydrase, catalytically similar

Carbonic anhydrase ings, Narumi & Miyamoto (1974) suggest the following sequence of events in the regulation of gastric acid secretion: (1) gastric stimulants activate gastric mucosal adenyl cylase resulting in an increase in cyclic AMP, (2) cyclic AMP activates a protein kinase which (3) phosphorylates and thus activates carbonic anhydrase. As yet this phosphorylation-activation phenomenon has been shown only for purified bovine erythrocyte carbonic anhydrase. The findings are most interesting and clearly call for further investigation. They imply a general significance for the regulation of carbonic anhydrase activity in organs of secretion. Different kinetic properties and tissue distribution of high and low activity carbonic anhydrase suggest that the isozymes may have different physiological functions (see Carter, 1972). This possible functional difference provocates further investigation of the quantitative distribution of the isoenzymes in a wide variety of tissues. There is strong evidence that such investigations should not overlook the possibility of membrane bound carbonic anhydrase. Relatively few studies have included an examination of the subcelluCarbonic anhydrase in other vertebrate tissues lar distribution of carbonic anhydrase. Studies prior A comprehensive list of animal tissues known to to 1972 have been reviewed by Carter (1972). Recently contain carbonic anhydrase is beyond the scope of a most interesting phenomenon has been observed this review. The work prior to 1972 has been thor- by Kimura & MacLeod (1975) during an investigaoughly discussed by both March (1967), and Carter tion of the involvement of carbonic anhydrase in the (1972). We limit ourselves here to some of the more secretion of hormones from the anterior pituitary recent findings which indicate directions for future gland of rats. About 70~ of the pituitary carbonic research. anhydrase was found to be membrane bound, the Early studies which indicated that the carbonic remainder being in the soluble fraction. Electroanhydrase in bone tissue is confined to preerythro- phoretic and immunologic analyses revealed that the cytes led to the conclusion that the enzyme does not particulate enzyme is a low activity carbonic anhydplay a role in bone mineralization or demineralization rase, and the soluble enzyme a high activity isozyme. (see Maren, 1967). However, Minkin & Jennings During in vitro incubation of pituitary glands, se(1972) have shown that sulfonamide inhibitors of car- cretion of prolactin was accompanied by a shift of bonic anhydrase inhibit the parathormone-induced the low activity isozyme from the particulate to the resorbtion of bone in organ culture. More recently soluble fraction. Both prolactin release and solubilizaGay & Mueller (1974), using autoradiography after tion of the particulate enzyme were inhibited when administration of tritiated acetazoleamide, have pituitaries were incubated in the presence of dopademonstrated what appear to be significant quantities mine. A relationship between the observed shift and of carbonic anhydrase in osteoclasts from quail and the release of hormone is suggested. The findings of chickens. These findings reopen the question of the Kimura & MacLeod raise interesting questions role of carbonic anhydrase in bone metabolism, and regarding the nature of the carbonic anhydrase bindprovide at least preliminary evidence that the enzyme ing sites, the physiological significance of the shift plays a role in bone resorption. from the binding sites to the cytoplasm, and whether Gastric mucosa contains relatively large amounts this phenomenon occurs in other secretory tissue. of carbonic anhydrase. The suggestion that its general In another recent study McKinley & Whitney function is to catalyze CO2 hydration and thus pro- (1976) have found that about 2~o of human kidney vide hydrogen ions for secretion is supported by the carbonic anhydrase appears in particulate fractions, finding that acetazoleamide inhibits the secretion of apparently bound to brush border membranes. If the hydrogen ions (see Carter, 1972). However, its signifi- enzyme is concentrated at specific sites in the kidney, cance for gastric acid secretion has not been definitely it could have a local catalytic effectiveness of much established. There are some recent findings which greater significance than indicated by its simple promay reveal a clearer picture of the role of carbonic portion of total kidney carbonic anhydrase activity. anhydrase in the process of gastric secretion. Gastric It is suggested that it may be localized at membrane acid stimulants such as gastrin and histamine, as well sites of bicarbonate reabsorption. Characterization of as cyclic AMP, have been found to stimulate the car- this particulate carbonic anhydrase has not been combonic anhydrase activity of gastric tissue in rats (Nar- pleted. In its susceptibility to inhibition by anions it umi & Kanno, 1973; Narumi & Maki, 1973). Subse- resembles the high activity erythrocyte isozyme. Howquently, Narumi & Miyamoto (1974) have shown that ever, in contrast to the erythrocyte enzyme, it is stable purified bovine erythrocyte carbonic anhydrase is in the presence of dodecyl sulfate and when high acactivated by cyclic AMP-dependent protein kinase, tivity erythrocyte enzyme was added to kidney memand that the activation is associated with phosphoryl- brane preparations, no significant binding was ation of the carbonic anhydrase. Based on these find- observed. Soluble, cytoplasmic kidney carbonic

to the mammalian high activity isozyme. This would seem to lend support to the suggestion that the existence of the two major isozymes is the result of gene duplication, and that, with respect to evolution, the high activity isozyme represents the older molecule (Ashworth et al., 1971; Tashian et al., 1975). From a continuing study of the evolutionary history of carbonic anhydrase isozymes, Tashian et aL (1975) have suggested that the proposed gene duplication took place early in mammalian evolution. They note that preliminary examination of a marsupial (red kangaroo, Marcropus rufus) showed only high activity carbonic anhydrase in erythrocytes. This suggests, if confirmed for other marsupials, that the duplication may have occurred after the divergence of placental mammals (about i00 million years ago). However, as we have mentioned previously (see also Carter, 1972) the absence of the low activity isozyme from erythrocytes does not mean that it is not present in other tissues. To our knowledge, there has been no report of attempts to identify carbonic anhydrase isozymes in other tissues of the nonmammalian vertebrates.

4

HALLIEF. BUNDY

anhydrase has been shown to be identical to the high activity erythrocyte isozyme (Wistrand et al., 1975). The results of these studies imply a functional difference between soluble and particulate carbonic anhydrase. Further study of the subcellular distribution of the enzyme, the characterization of soluble and particulate forms, and their relation to high and low activity erythrocyte isozymes has gained in importance as a result of these findings. Carbonic anhydrase in development It has long been known that the carbonic anhydrase activity of erythrocytes and other tissues of several vertebrate species increases during development from the embryo to the adult organism (see Maren, 1967). However most of the work was done before the discovery of low and high activity forms of the enzyme and the activity measured was presumably due to a mixture of the isozymes. Evidence suggesting different functions for the two isozymes, and that they are products of separate structural genes (see Tashian et al., 1975) raises the question of different rates of appearance and the possible adaptive significance of the isozymes during development. Preliminary experiments in a study of carbonic anhydrase in amphibian metamorphosis have shown that for R. catesbeiana blood the carbonic anhydrase activity per erythrocyte in prometamorphic tadpoles is about 20 x less than in the adult frog (Bundy & Cheng, 1976). Thus it is apparent that a large increase in erythrocyte carbonic anhydrase activity occurs during the development of the prometamorphic tadpole to the adult frog. Whether this increase is the result of increased synthesis or due to a shift in synthesis from a low activity to a high activity enzyme remains to be determined. In either case the change may have adaptive significance. Among the many adaptive changes which take place during the transition of anurans from an aquatic larval form to an essentially terrestial adult, is the shift from tadpole to adult hemoglobin (reviewed by Frieden & Just, 1970). It has been suggested that the primary role of erythrocyte carbonic anhydrase may be to allow an efficient utilization of the Bohr effect in the unloading of oxygen by hemoglobin (Schmidt-Nielsen, 1970). Since tadpole hemoglobin does not exhibit an significant Bohr effect (Aggarwall & Riggs, 1969), carbonic anhydrase would be of little value in facilitating oxygen unloading. However, adult frog hemoglobin has a marked Bohr effect and carbonic anhydrase could be of significance in facilitating the unloading of oxygen during the short time the blood remains in the capillaries. There is evidence that in the developing tadpole the site of red blood cell formation shifts from kidney to liver, and after metamorphosis, to bone marrow (Broyles & Frieden, 1973; Watt & Riggs, 1975). It would be of interest to study the carbonic anhydrase associated with each of these distinct red cell populations. There is some evidence to suggest that in humans, erythrocyte carbonic anhydrase activity may be related to the shift from synthesis of fetal hemoglobin to synthesis of adult hemoglobin. Eng & Tarail (1966) described a syndrome in which the erythrocytes of an adult patient were characterized by low carbonic anhydrase activity, high HbF, and low HbA2, a situ-

ation found normally in the newborn infant. Electrophoresis of hemolysates followed by staining with ~-naphthyl acetate indicated a deficiency of both high and low activity isozymes. However, recently developed immunological techniques (see for example Anker & Mondrup, 1974) offer a more specific and quantitative method for the detection and measurement of carbonic anhydrase isozymes in complex mixtures such as red cell hemolysates or tissue homogenates. The biosynthesis of carbonic anhydrases should prove to be a particularly fruitful area of investigation. Desimone et al. (1973) have described methods for studying the biosynthesis of both high and low activity carbonic anhydrases in reticulocytes and bone marrow erythroid cells of the pig-tailed macaque, Macaca nemestrina. Bone marrow cultures might offer possibilities for further investigation of carbonic anhydrase biosynthesis, especially with regard to gene activation and inactivation. Barker et al. (1973) have recently described a method of preparing and culturing bone marrow cells for the study of hemoglobin switching in goats. The use of these or similar techniques may provide answers to such questions as the relationship between carbonic anhydrase and the fetal pattern for hemoglobin, and Anker & Mondrup's (1974) suggestion that thyroxine acts as a regulator of low activity carbonic anhydrase in erythroid marrow. INVERTEBRATE CARBONIC ANHYDRASE

Because of its widespread occurrence throughout the plant and animal kingdoms, carbonic anhydrase would seem to be one of those appropriate biomolecules with which to attempt, in the words of Florkin (1973), "to define descent with change at the molecular level." However, despite its detection in various tissues of most major classes of invertebrates, (see Maren, 1967) work on the purification and characterization of invertebrate carbonic anhydrase has lagged far behind study of the vertebrate enzymes. Addink (1971) has partially purified a carbonic anhydrase from mantle muscle of the cephalopod mollusc Sepia officinalis. The enzyme is reported to resemble the high activity mammalian erythrocyte isozyme in its catalysis of COz hydration and inhibition by sulfonamides. However, the solubility characteristics of this cephalopod carbonic anhydrase are quite different from those of the vertebrate erythrocyte enzymes. Initial extracts were turbid even though Triton X-100 was employed, and the acetone powder from these extracts was insoluble in aqueous buffers. Deoxycholate was used for solubilization of the final preparation. The most thoroughly characterized invertebrate carbonic anhydrase to date is that obtained from oyster (Crassostrea virginica) serum (Nielsen & Frieden, 1972). Carbonic anhydrase apparently exists in oyster serum as a high molecular weight (26.6S) aggregate. Disaggregation of partially purified oyster carbonic anhydrase by polyacrylamide gel electrophoresis yielded a homogeneous preparation with an apparent molecular weight of about 500,000 (13.6S). This may also be an aggregate. Preliminary Zn analysis indicated 13Zn/molecule based on 500,000MW. Con-

Carbonic anhydrase ceivably the preparation obtained could be an aggregate of 13 subunits each having a molecular weight of 38,000 (about the size of the elasmobranch erythrocyte enzyme) and containing 1 zinc ion. The activity of the 26.6S aggregate increased by 35% in the presence of dithiothreitol, a sulfhydril reducing agent. Since this was accompanied by only a slight loss in turbidity, it was concluded that the increase in activity was not due to disaggregation. The effect of dithiothreitol on the final 13.6S preparation was not reported nor were pCMB-reactive-SH groups determined. Further information regarding these effects will be of interest, especially in view of the high cysteine content of elasmobranch carbonic anhydrase, and the essential SH groups in both the amphibian and avian enzymes. Carbonic anhydrase appears to play a role in CaCO3 deposition in molluscan shell formation (see Wilbur, 1972). There is also evidence for its participation in a specialized process of decalcification which allows certain molluscs to penetrate the shells of bivalves. Chetail & Fourni6 (1969) found carbonic anhydrase activity in homogenates of the accessory boring organ from the nuricid gastropod Purpura lapillus. In in vitro experiments the shell-boring activity of these gastropods appears to be inhibited by acetazoleamide. We have found that boring organs from the naticid gastropod Polinices lewisii contain a substantial amount of carbonic anhydrase. The activity per mg protein is 3 x that from mantle tissue, and 6 x that from gill tissue (Bundy, unpublished results). Preliminary experiments with a partially purified preparation of this enzyme indicate that like vertebrate carbonic anhydrases it catalyzes the hydrolysis of p-nitrophenylacetate as well as the hydration of CO2, and is inhibited by acetazoleamide. Unlike the vertebrate enzymes, it appears to have a molecular weight > 100,000. The latter finding, and similar indications for Sepia mantle, and oyster serum carbonic anhydrases, suggest that existence as high molecular weight aggregates may be a common feature of invertebrate carbonic anhydrases. It seems likely that carbonic anhydrase will be found in other organisms, such as the clionid sponges, which are capable of penetrating calcareous substrates. In view of the previously mentioned experiments on bone resorption, it appears that carbonic anhydrase has a long evolutionary history of involvement in natural processes of calcification and decalcification, extending from the lower invertebrates to man.

P L A N T CARBONIC ANHYDRASE

While considerably more attention has been devoted to carbonic anhydrase in animals, significant advances have recently been made in the investigation of plant carbonic anhydrase. The early interest in plant carbonic anhydrase, stemming from its possible role in photosynthesis, may have been dampened somewhat by the report that the enzyme is not associated with chloroplasts (Waygood & Clandenning, 1950). The observations of Everson & Slack (1968) resulted in a resurgence of interest in plant carbonic anhydrase. They found that up to 67% of total leaf

carbonic anhydrase is localized in the chloroplasts of plants, such as spinach and peas, which photosynthesize via the Calvin cycle. However, plants such as maize, which fix CO2 into C4 dicarboxylic acids via phosphopyruvate carboxylase, were characterized by a much lower level of carbonic anhydrase activity which appeared to be cytoplasmic rather than chloroplastic. Poincelot (1972a) confirmed that about 63% of spinach leaf carbonic anhydrase is present in the chloroplasts where, like ribulose diphosphate carboxylase, it is associated with the stroma. However, the previous evidence regarding a relationship between the type of CO2 fixation characteristic of the plant, and its carbonic anhydrase localization and content may require reevaluation. Poincelot (1972b), using improved grinding techniques in the presence of an -SH protecting reagent and N2, found the total carbonic anhydrase activity of maize to be comparable to that in spinach, and that the bulk of the enzyme is located in mesophyl chloroplasts. Ribulose diphosphate carboxylase was found to be about equally distributed between mesophyl and bundle sheath cells. While there is considerable evidence that carbonic anhydrase participates in photosynthesis, its exact role remains an open question. Three hypotheses regarding its possible function are discussed by Everson (1971), Graham & Reed (1971), and Poincelot (1972b). Very briefly these are: (1) Carbonic anhydrase, in close proximity to ribulose phosphate carboxylase, may provide the CO2 for maximal rates of CO2 fixation by the latter enzyme; (2) it may facilitate the transfer of COa across the chloroplast membrane; and (3) it may act by catalyzing the hydration of CO2 thus providing protons for the maintainance of a proton gradient which is assumed to regulate photophosphorylation. The revival of interest in plant carbonic anhydrase has stimulated attempts to isolate and characterize the enzyme in order to provide a molecular basis for its apparent regulatory role in photosynthesis. There are indications that plants as well as animals contain isozymic forms of carbonic anhydrase. The results of polyacrylamide gradient gel electrophoresis (a technique in which separation is dependent primarily on molecular size and shape) of leaf extracts from 24 species of flowering plants has revealed two main types of plant carbonic anhydrase: low mobility and high mobility forms (Atkins et al., 1972a). Most interesting was the finding that in most species examined each type occurred in pairs; the high mobility pairs found principally in monocotyledons, and the low mobility pairs in dicotyledons. Atkins et al. (1972b) purified carbonic anhydrase from pea leaves (P. satirum, a dicotyledon) and from Tradescantia albiflora K. (a monocotyledon). As expected on the basis of their previous electrophoretic studies, the enzymes differ significantly in molecular size. The apparent molecular weights from gel filtration are 42,000 for the Tradescantia enzyme, and 188,000 for the pea leaf enzyme. Each purified preparation consisted of a pair of carbonic anhydrase isozymes corresponding in mobility to those found in the crude leaf extracts. More recently Kachru & Anderson (1974) have reported the Separation of chloroplastic and cytoplasmic forms of pea leaf carbonic anhydrase by isoelec-

6

HALLIEF. BUNDY

tric focusing. The isozyme pairs observed by Atkins et al. may indeed be chloroplastic and cytoplasmic forms of the enzyme. To date the most highly purified and thoroughly characterized preparations of plant carbonic anhydrase have been obtained from the dicotyledons: parsley (Tobin, 1970), pea leaves (Kisiel & Graf, 1972), and spinach (Pocker & Ng, 1973). In each case the molecular weight is about 180,000, with 6 tightly bound zinc ions per molecule. A major difference between these enzymes and vertebrate erythrocyte carbonic anhydrases is that the former are hexameric, consisting of 6 subunits. Each subunit is apparently similar in size and zinc content to the erythrocyte enzymes. There are other striking differences between plant and animal carbonic anhydrase. Acetazoleamide is only a moderate inhibitor of the plant enzyme (Ki > 10 -5 M, Pocker & Ng, 1974), and the plant enzymes lack the catalytic versatility of the vertebrate erythrocyte carbonic anhydrases. None of the plant carbonic anhydrases examined catalyzes the hydrolysis of nitrophenyl acetates. On the basis of these differences, Pocker & Ng (1974) have suggested that the active site crevice of plant carbonic anhydrase is considerably smaller than that of the erythrocyte enzyme. Although the actual number of active sites in the hexameric plant enzyme has not been determined, Pocker & Ng (1974) have found that it does not appear to be an allosteric enzyme. Experimental data indicates that each active site acts independently, forming a 1:1 complex with the substrate, CO2 or with the inhibitors, acetazoleamide and azide. Like the elasmobranch, amphibian, and avian carbonic anhydrases, the plant enzymes have a relatively high cysteine content. The state of the -SH groups is not clear. Parsley carbonic anhydrase loses activity in the absence of a sulfhydril reducing agent, and is reversibly inhibited by pCMB (Tobin, 1970). However, the spinach enzyme does not require a sulfhydril reducing agent for stability, and reacts with a specific -SH reagent, 5,5' dithiobis(2-nitrobenzoate), only after dissociation of the hexamer in 6 M guanidine hydrochloride (Pocker & Ng, 1973). In contrast to the findings of Atkins et al. (1972a, b), isozymic forms were not detected in the purified preparations of parsley (Tobin, 1970), pea leaf (Kisiel & Graf, 1972) or spinach (Pocker & Ng, 1973) carbonic anhydrase. However, the latter investigators did not use the polyacrylamide gradient gel electrophoresis employed by Atkins et al. Further investigation of what appears to be a fundamental difference between monocotyledon and dicotyledon carbonic anhydrase, of the relationship between cytoplasmic and chloroplastic carbonic anhydrases, and of the effects of conditions such as COz concentration and light intensity on the biosynthesis of these enzymes, are expected to provide informarion of value for the ellucidation of the physiological role or roles of carbonic anhydrase in plants.

REFERENCES

ADDINKA. D. F. (1971) Carbonic anhydrase of Sepia officinalis L. Comp. Biochem. Physiol. 38113,707-721. ADLER L., BRUNDELLJ.. FALKBR1NGS. O. & NYMAN P.

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