discovery of carbonic anhydrase in rabbit skeletal muscle and evidence for its identity with

Discovery of Carbonic Anhydrase in Rabbit Skeletal Muscle and Evidence for Its Identity with “Basic Muscle Protein”*

(Received for publication, November 21, 1977)

Adele M. Register, Martha K. Koester, and Ernst A. NoRmann+

From the Dewartment of Biochemistrv and the Prom-am in Biomedical Sciences, University of California, Riverside, California 92521 ’

.,

“Basic Muscle Protein,” a protein with unknown function that had previously been isolated in homogeneous form and partially characterized while obtained as a by-product of the purification procedure for phosphoglucose isomerase (Blackburn, M. N., Chirgwin, J. M., James, G. T., Kempe, T. D., Parsons, T. F., Register, A. M., Schnackerz, K. D., and Noltmann, E. A. (1972) J. Biol. Chem. 247, 1170-1179), has been shown to be identical with carbonic anhydrase. This identification is based on its activity as a CO2 hydratase (which is resistant to inhibition by sulfonamide), its zinc content (1 g atom/monomer), and the stoichiometry of the loss of its activity that accompanies removal of zinc.

Based on its amino acid composition, its tryptic peptide maps obtained with or without carbamylation, and its immunochemical properties, as compared with those same parameters determined for rabbit erythrocyte carbonic anhydrases I and II, this new carbonic anhydrase isoenzyme from muscle is proposed to derive from a third genetic locus and is designated as carbonic anhydrase III.

Carbonic anhydrase III constitutes 1 to 2% of the total cytosol proteins extracted at low ionic strength from rabbit skeletal muscle. As shown by ultracentrifuga- tion, electrophoresis, and gel filtration, the enzyme ex- ists in both monomer (s&, = 3.2 S; M, = 29,000) and dimer (&,,~ = 4.7 S; M, = 58,000) forms. The monomer- dimer equilibrium is dependent on the state of sulfhy- dry1 oxidation in that the dimer has been found to contain 12 total half-cystine residues, 10 of which are present as free sulfhydryl groups, whereas the monomer contains 6 total half-cystine residues, all of which can be titrated withp-mercuribenzoate. These data are taken to suggest that the dimer is produced by formation of an intermolecular disulfide bridge between two monomers.

Carbonic anhydrase III is proposed to function in muscle as a buffer system that participates in regulat- ing intracellular CO2 concentration and pH by maintaining the equilibrium between COZ and HCOs-.

Since the discovery of carbonic anhydrase in 1932 (l), it has become one of the best characterized of all enzymes. Work

* This work was supported in part by United States Public Health Service Research Grant AM 07203 and Biomedical Research Devel- opment Grant RR 09070. Part of the investigations described here are from a dissertation to be submitted by M. K. K. to the Graduate Division of the University of California, Riverside, in fulfillment of the requirements for the degree of doctor of philosophy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Y$ To whom inquiries about this paper should be addressed.

concerning its structure and mechanism (2, 3), genetic rele- vance (4-6), and physiological function (7, 8) has been exten- sively reviewed. In mammals, the enzyme has two genetically distinct forms which are designated as carbonic anhydrase I (the low activity form) and carbonic anhydrase II (the high activity form). The complete, or partially complete primary structures of several mammalian carbonic anhydrase I and II isoenzymes have now been determined, and the number of identical residues at homologous positions was found to range from 56 to 62% (6). The isoenzymes are immunochemically as well as genetically distinct from each other (9). In addition, there are many electrophoretic variants of each of the isoenzymes. All cross-react immunochemically with carbonic anhydrase I or II, regardless of whether they derive from point mutations or epigenetic modifications (10).

Our interest in this enzyme did not originate as a deliberate attempt to study carbonic anhydraseper se but was the result of several years of frustrating search for the identity of an unknown protein obtained in essentially homogeneous form as a by-product of what had then become our standard purification procedure for phosphoglucose isomerase (11). This “Peak X” (see Fig. 6A of Ref. 11) amounted to 1 to 2% of the total protein extractable from skeletal muscle at low ionic strength. In the absence of any clue as to the nature of this protein, studies on its physical and chemical properties, including tests for its homogeneity, were implemented to provide parameters for comparison with known proteins. It was deemed inconceivable at the time that a major component of the solubilizable protein of skeletal muscle had not yet been identified. Quantitative hydrodynamic studies yielded a molecular weight of approximately 30,000 and amino acid analyses indicated a rather high content of basic amino acids, tryptophan and cysteine, leading to its designation as “Basic Muscle Protein.” However, this protein did not bear a resem- blance to any of the enzymes involved in glycolysis. It is notable in this context that in 1966, Scopes (12) had reported the existence of an unknown protein (“Protein F”) that was separable from the major muscle cytosol proteins by electrophoresis and that appeared to have some properties similar to those found for Basic Muscle Protein. Protein F had also been found not to be active in any of the glycolytic enzyme assays or as an ATPase (12).

Comparison of the amino acid composition of Basic Muscle Protein with the amino acid content of other proteins (13) suggested some similarity to certain carbonic anhydrases. However, when assayed as a p-nitrophenylacetate esterase, it had minimal activity as compared with erythrocyte carbonic anhydrases 1 and II. Furthermore, concentrations of acetazolamide (a highly specific carbonic anhydrase inhibitor) which can totally inhibit the latter had no effect on this activity. This observation caused us temporarily to conclude that the similarity of its amino acid composition to those of other

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4144 Carbonic Anhydrase

carbonic anhydrases may be coincidental and to resume our search for other activities. However, three events in 1976 led us back in the right direction. 1) We came across reports in the literature describing carbonic anhydrase species relatively resistant to sulfonamide inhibition (e.g. Refs. 14 and 15). 2) Continuing characterization of Basic Muscle Protein yielded stoichiometric amounts of zinc as an integral part of the protein molecule. 3) A report was published by Holmes (16) in which he described the existence of basic protein bands with carbonic anhydrase activity in electrophoretic patterns of crude muscle extracts. Unfortunately, it was not clear from that communication whether this activity had been tested for sensitivity to sulfonamide inhibition, which is normally considered a mandatory criterion for identifying carbonic anhydrase in unpurified systems.

We then assayed Basic Muscle Protein as a COa hydratase and found it to have low (20% of carbonic anhydrase I), concentration-dependent activity that was resistant to acetazolamide inhibition. In addition, systematic zinc analyses indicated a direct relationship between removal of zinc and loss of COZ hydratase activity.’ Thus, we concluded that Basic Muscle Protein appeared to be a new, possibly muscle-specific, carbonic anhydrase controlled by a third genetic locus (17).

This conclusion is highly significant in view of the relatively large amounts that are present in muscle, a tissue that has traditionally been conspicuous for its absence from carbonic anhydrase tissue distribution lists (7, 8). In retrospect, this is

not surprising since an enzyme is normally purified by identifying its activity in crude tissue extract and by applying techniques of protein purification to select fractions with successively increasing specific activity. I f a situation prevails, therefore, that does not allow unequivocal identification of that enzyme activity because COZ hydratase activity that is not sulfonamide-resistant can be simulated by other metabolic processes that result in the production of H’, the existence of a low activity, sulfonamide-resistant carbonic anhydrase was, by definition, impossible to ascertain. It was only the reverse process, viz. the identification of such carbonic anhydrase activity as the property of an already isolated and characterized protein that made this discovery possible.

This paper will provide evidence that Basic Muscle Protein is more appropriately defined as carbonic anhydrase III and report some of its physical and chemical characteristics. A preliminary account of part of this work has been given previously ( 18).

EXPERIMENTAL PROCEDURES

Materials

Rabbit blood carbonic anhydrases I and II were prepared as described previously (19). Reagents for enzyme assays, enzyme purification, buffers, physical measurements, protein modification, amino acid analysis, and peptide mapping were of the highest grades com- mercially available. All solutions were made with deionized, glass- distilled water.

Methods

Protein Purification-The isolation procedure for carbonic anhydrase from rabbit skeletal muscle is based on our previously described method for obtaining phosphoglucose isomerase from the same tissue (11). The fractions containing carbonic anhydrase activity that elute from the carboxymethyl Sephadex column (10 to 40 mM sodium phosphate gradient, pH 6.90 * 0.02) are pooled and concentrated in an Amicon concentration cell holding a suitable Dia-Flo ultrafiltration membrane. The pool of carbonic anhydrase activity is then further purified by molecular sieve chromatography on a Sephadex G-75 (2.5 x 90 cm) column equilibrated with 100 mrvr sodium phosphate buffer,

’ Results of these analyses have been reported in detail in our previous communication (17).

III of Rabbit Muscle

pH 6.90. The carbonic anhydrase fractions, which elute as a symmetrical peak, are pooled, concentrated as described above, precipi- tated with ammonium sulfate, and stored as a frozen paste.

All preparative procedures are conducted at 4°C and, throughout all fractionation steps, 1 mM dithiothreitol is present.

Enzyme Assays-Carbonic anhydrase activity was determined by the modification of the Wilbur-Anderson calorimetric method (20) introduced by Rickli et al. (21), as previously used in this laboratory (17, 19). One unit of enzyme activity is defined as 1 pmol of H’ produced/liter/s at 4”C, the titration equivalents of H’ being calculated in a manner analogous to that used by Maren et al. (22).

Protein Measurements-During the isolation of carbonic anhydrase, protein concentrations were determined by the calorimetric biuret method of Gornall et al. (23), after first precipitating the protein with 10% trichloroacetic acid. A factor of 16 mg of protein/absorbance unit was employed for a 5-ml total volume, measured at 540 nm in a l-cm light path. Protein concentrations of the homogeneous enzyme were measured either by a scaled down version of the biuret method or by direct measurement of the protein absorbance at 280 nm with use of an extinction coefficient of 2.32 for a 0.1% solution in 100 mM sodium phosphate buffer, pH 6.90 (see “Results”).

Zinc Analysis-Quantitative zinc determinations were made with the aid of a Perkin-Elmer model 303 atomic absorption spectropho- tometer by absorptivity measurements at 2138 A, according to the procedure outlined in the Perkin-Elmer manual (24). Analyses were performed in duplicate with all readings taken against blanks containing buffer in place of dialyzed protein solutions. The stoichiometry of the zinc content was determined by five readings at five different protein concentrations.

Amino Acid Analyses-Enzyme aliquots were hydrolyzed for 20 h at 110 + 0.2”C in 6 N HCl and analyzed for their amino acid contents by standard procedures employed in this laboratory, as previously described in detail (25). A separate hydrolysis time study (20-, 40-, and 70-h time points) was made to determine maximal recovery or extrapolation to zero time hydrolysis for the residues so affected. Tryptophan was determined according to the method of Edelhoch (26). Total half-cystine was measured as cysteic acid after performic acid oxidation following the method of Moore (27). Sulfhydryl assays were performed by the p-mercuribenzoate method of Boyer (28) as previously used in this laboratory (25) with the addition of 0.5% sodium dodecyl sulfate in order to facilitate obtaining a well defined end point.

Peptide Mapping-Peptide maps were obtained by modification of the method of Katz et al. (29) as previously described in detail (30). Prior to the tryptic digestions, enzyme samples were routinely carboxymethylated in 8 M guanidine hydrochloride at pH 8.2 with iodoacetate (or with iodo[?]acetate), following the method of Crest- field et al. (31). When only arginine peptides were desired, carbamylation was performed in addition to the carboxymethylation by reaction of the enzyme with potassium cyanate in 6 M guanidine hydrochloride, 2 M N-ethylmorpholine (pH 8.1) according to Stark (32). Ascending chromatography in 1-butanol:acetic acid:water (4:1:5, v/v) was performed first, followed by electrophoresis at pH 3.5 (pyridine:acetic acid:water, 1:10:289, v/v) for which a constant potential of 700 V was applied at right angle to the direction of chromatography. Peptides labeled by carboxymethylation with iodo- [14C]acetate were visualized by autoradiography (14 to 21 days of exposure). Tryptophan peptides were identified by Ehrlich’s reagent (33). Arginine peptides were localized under ultraviolet light after staining with phenanthrenequinone (34) and peptides with free NHZ- terminal residues by spraying with fluorescamine and observation also under ultraviolet light.

Polyacrylamide Gel Electrophoresis-Cylindrical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed by a modification of the method of Weber and Osborn (35), as previously described (30). Gel concentrations of 10% were employed throughout. Slab gel electrophoresis in a Studier apparatus (36) was also performed in the presence of sodium dodecyl sulfate. Stacking gels were 5% and the resolving gels were either 9 or 12%.

Gel Filtration-When molecular sieve chromatography was used for the purpose of molecular weight estimations, a Sephadex G-75 column analogous to that described for the final purification (2.5 x 80 cm) was employed which was calibrated with appropriate standard proteins of known molecular weight. In each case, the freshly equilibrated column was eluted with 100 mM sodium phosphate buffer, pH 6.90 at 4”C, and fractions of constant volume were collected. Molec- ular weights were estimated from a plot of K,, versus log molecular weight, with K,J being equivalent to ( V, - Vo)/V,.

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Carbonic Anhydrase III of Rabbit Muscle

5 COLUMN EFFLUENT (ml)

FIN. 1. Chromatography on CM-Sephadex of Basic Muscle Pro- tein according to the method developed for the purification of rabbit muscle phosphoglucose isomerase. For details, refer to “Results.”

Isoelectric Focusing-Isoelectric points of the carbonic anhydrase monomer and dimer forms were determined by preparative isoelectric focusing in an LKB model 8101 focusing column (capacity, 110 ml) by applying a constant potential of 600 V for 96 to 120 h at 4°C. Sucrose density gradients containing LKB Ampholines or Brinkmann pHisolytes in the pH range from 8 to 10 were prepared with an LKB model 8121 gradient mixer. Fractions of 1 ml were collected at a flow rate of 30 ml/h and assayed for pH, protein concentration, and CO2 hydratase activity.

Ultracentrifugal Analyses-Sedimentation studies were conducted with the aid of a Beckman model E analytical ultracentrifuge equipped with an RTIC unit and a photoelectric scanner. Absorbance measurements at 280 nm were made directly from the scanner print- out according to recommended procedures (37). Carbonic anhydrase monomer samples were dialyzed to equilibrium against 15 mM dithiothreitol, 100 mM sodium phosphate buffer, pH 6.9; carbonic anhydrase dimer samples were dialyzed against the same phosphate buffer without, dithiothreitol. All runs were made in an An-D rotor with a filled Epon 2.5” double sector centerpiece and sapphire windows. Sedimentation velocity runs were performed at 10°C at 59,780 rpm. Apparent sedimentation coefficients were calculated from the rate of boundary migration and corrected to standard conditions according to accepted methodology (38). Sedimentation equilibrium ultracen- trifugation experiments followed the meniscus depletion method of Yphantis (39) and Van Holde (40). A partial specific volume of 0.731 as calculated from the total amino acid composition was used in all computations (41).

Purification of Carbonic Anhydrase III from Rabbit

Skeletal Muscle

As referred to in the introduction, the purification of car-

bonic anhydrase from rabbit skeletal muscle was developed

as purification of Basic Muscle Protein incidental to the

isolation procedure for phosphoglucose isomerase (11). Fig. 1,

reproduced in modified form from our earlier paper (ll), shows the elution pattern from a CM-Sephadex column as

originally observed for the phosphoglucose isomerase purifi-

cation procedure in which Basic Muscle Protein elutes at 36

mM sodium phosphate as compared with 25 mM for phospho-

glucose isomerase. Fig. 2 represents the elution profile ob-

tained on rechromatography on Sephadex G-75 of the pooled

fractions of Basic Muscle Protein from the CM-Sephadex column. It is evident that, in the presence of 1 mM dithiothreitol, carbonic anhydrase elutes as a single symmetrical peak in this chromatographic system, well resolved from some contaminating proteins of higher molecular weight. The material appears to be homogeneous also on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as shown in Fig. 3. In terms of protein weight, approximately 1.5 times as much carbonic anhydrase is obtained in this procedure as compared with phosphoglucose isomerase. Based upon the original muscle extract, this amount corresponds to approximately 1 to 2% of the total protein solubilized by extraction with 0.01 M KCl.

FRACTION NUMBER FIG. 2. Chromatography of Basic Muscle Protein (carbonic anhy-

drase III) on Sephadex G-75. 0- --0, protein concentration; U, CO, hydratase activity; V---V, zinc content. For details, refer to “Methods.”

Occurrence of Carbonic Anhydrase III as Both Monomer

and Dimer Species

I f the isolation of carbonic anhydrase III was not conducted in the presence of a reducing agent, an additional peak appeared during Sephadex G-75 chromatography just prior to the considerably larger peak (Fig. 4A, peak volume, 210 ml). (This is not to be confused with non-carbonic anhydrase proteins eluting at 186 ml preceding the high molecular weight carbonic anhydrase species.) On rechromatography under identical conditions, this small peak was also found to be essentially homogeneous (Fig. 4B). The peak at 210 ml was found to have essentially the same amino acid composition as compared with the material eluting in the peak at 260 ml. Subsequent rechromatography of the former on Sephadex G- 75, calibrated with protein standards of known molecular weight, suggested that the protein species eluting at 210 ml is a dimer of the material eluting at 260 ml. This was eventually confirmed by ultracentrifugal analysis (see below).

Since the relative amounts of monomer and dimer appeared to be affected by the presence of reducing agent, their interconversion was studied in more detail. When an aliquot of Pool M-l (Fig. 4A) was rechromatographed in the absence of dithiothreitol, two peaks appeared, one in the monomer position and one in the dimer position (Fig. 4C). Because more dimer was present than was expected on the basis of their relative quantities in the preceding chromatographic step, it was assumed that production of dimer occurred at the expense of monomer. A second aliquot of Pool M-l was then rechromatographed after having been exposed for a period of 2 weeks to air oxidation (Fig. 40). This resulted in further change of the equilibrium between the species in the direction of the dimer, confirming that oxidative conditions favored dimer formation. We have never been able, however, to obtain physiological oxidizing conditions rigorous enough to completely convert all of the monomer to dimer. In contrast, it is possible to completely convert the dimer (Pool D-2 of Fig. 40) to the monomer by exposing it for 1 week to 10 mM

dithiothreitol followed by chromatography in the presence of a 1 mM concentration of the reducing agent (Fig. 4E)

General Physical Parameters

Sedimentation Coefficients-Apparent sedimentation coefficients (szgy(,) varied in linear fashion with protein concentrations as shown in Fig. 5 for both the monomer and the dimer species. At all concentrations of both species, the boundary moved as a sharp symmetrical peak in the schlieren optical system during the entire time of the run. This finding suggests

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FIG. 3. Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate of fractions obtained during the purification of rabbit muscle carbonic anhydrase III. Crude Basic Muscle Protein from CM-Sephadex column (see Fig. 1); Sephadez G- 75 Void Volume Proteins from Sephadex G-75 column, eluant peak at 186 ml (see Fig. 4A); CA III Monomer Eluted from Sephadex G-75, eluant peak at 260 ml (see Fig. 4A).

that there was no interconversion of monomer and dimer species during the sedimentation velocity experiments. Un- weighted least squares analysis of the data extrapolates to s&,~ values of 3.2 and 4.7 S for the monomer and the dimer, respectively. A sedimentation coefficient of 3.2 S for the monomer is within the range (2.7 to 3.3 S) previously reported for other carbonic anhydrases (2, 3) and is indicative of hydrodynamic properties for carbonic anhydrase III similar to those of carbonic anhydrases I and II. An ~$0,~ value of 4.7 S for the dimer is in an appropriate range for a globular protein of corresponding molecular weight.

Molecular Weight by Gel Filtration-An initial gross esti- mate of the molecular weights of both monomer and dimer species was obtained by molecular sieve chromatography on Sephadex G-75. Depending upon the chromatographic conditions and the protein standards used, molecular weights of 27,000 f 4,000 and 52,000 rfr 9,000 were observed for the monomer and dimer, respectively. Although a definite value can obviously not be derived from these data, it was evident in each instance that the dimer eluted at a volume reflecting approximately twice the molecular weight of that of the monomer. The large range of molecular weights obtained by this method illustrates the lack of reliability of this technique.

Molecular Weight by Sodium Dodecyl Sulfate-Polyacryl- amide Gel Electrophoresis-The molecular weight of rabbit muscle carbonic anhydrase III in the presence of sodium dodecyl sulfate has previously been reported (17). The rabbit muscle enzyme was found to migrate slightly more rapidly than either of the carbonic anhydrases from rabbit erythrocytes, perhaps reflecting its highly basic nature (42). In the sodium dodecyl sulfate gel electrophoresis system, the molecular weight was found to be 28,000 + 500, irrespective of whether the material analyzed was originally in the monomer or the dimer configuration.

Molecular Weight by Sedimentation Equilibrium Ultra- centrifugation-The weight average molecular weights determined by high speed equilibrium sedimentation (see “Meth- ods”) were found to be 29,000 + 500 for the monomer and 56,000 + 900 for the dimer. Fig. 6 shows typical data plots, their linearities suggesting no evidence for significant contam- inations with impurities of other molecular weights. To avoid formation of dimer from the monomer, all sedimentation

04 E 02 c g 12 C-J IO

0" 00 06 04 02

0.6 04 0.2

~WITHWI DTT,

02 .I5

0. I WITH 05

IO 20 30 40 50 60 70 80 90 100

FRACTION NO. FIG. 4. Chromatography on Sephadex G-75 of oxidized and re-

duced forms of carbonic anhydrase III from rabbit muscle. All samples were chromatographed on the same column (2.5 X 79 cm). A buffer flow rate of 24 ml/h and a time setting of 12.5 min/fraction were employed. The elution buffer was 0.1 M sodium phosphate, pH 6.9; the temperature was maintained at 4°C. Note the excellent reproducibility of the runs as evidenced by the essentially identical elution volumes for peak fractions indicated in the figure. The identities of the chromatographed samples are indicated on the right hand side of each section. A, 225 mg of dialyzed concentrate from CM-Sephadex chromatography; B, second rechromatography of Pool D-l (16 mg) from A; C, rechromatography of an aliquot (22.5 mg) of Pool M-l from A; D, rechromatography of another aliquot (22.2 mg) of Pool M-l from A after exposure to oxygen for 2 weeks; E, rechromatography of Pool D-2 (4.7 mg) from D after treatment with 10 mM dithiothreitol (0%“) for 1 week. Identification as monomer or dimer in the figure is to indicate that these peaks elute at volumes at which either monomer or dimer appear in a column calibrated with samples that have been so identified and characterized (see text).

equilibrium runs of the latter were performed in buffer containing 15 mM dithiothreitol. The molecular weight data and other general properties of carbonic anhydrase III are summarized in Table I. Based upon the number of analyses performed and the reproducibility of the data obtained, a value of 29,000 is considered to be the best approximation for the molecular weight of the monomer. Also, since the monomer is the principal molecular species and all of the available information points toward the dimer consisting of two identical monomers, the best value for the molecular weight of the dimer is assumed to be 58,000 (i.e. 2 x 29,000).

Isoelectric Focusing-The isoelectric points were found to be at pH 8.41 for the monomer and at pH 9.34 for the dimer. These values may be compared with a range of from pH 4.50 to 8.12 compiled in a recent review for carbonic anhydrases of various origins (43). This survey, however, did not include the values of 8.52, 9.00, and 9.63, respectively, for three sub forms of horse blood carbonic anhydrase II (44), which resemble more closely the highly basic values for rabbit muscle carbonic anhydrase observed by us.

Chemical Parameters

UV Protein Factor-An average extinction coefficient of

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Carbonic Anhydrase III of Rabbit Muscle 4147

I I I

m

/s:O,W =3.2S

J n u n

3.0 A

u

T I I I I T 0.1 0.2 0.3 0.4

INITIAL PROTEIN CONCENTRATION (mg’ml-‘1

FIN:. 5. Sedimentation coefficients of rabbit muscle carbonic anhydrase monomer and dimer forms as a function of protein concentration. For conditions, refer to “Methods.“’

RUN # 1139 MOLECULAR WEIGHT

RUN # 1135 MOLECULAR WEIGHT

Y I I I YI I I 49 50 51 49 50 51

r2 (cm21 r2 km2)

FIN. 6. Examples of high speed sedimentation equilibrium ultra- centrifugation runs of rabbit muscle carbonic anhydrase monomer and dimer species. A, monomer (0.20 mg/ml) centrifuged at 29,500 rpm; d(ln c)/d(r’) = 1.499. B, dimer (0.35 mg/ml) centrifuged at 21,740 rpm; d(ln c)/d(r’) = 1.549. For other conditions, refer to “Methods.”

2.32 at 280 nm was determined for a 0.1% solution of carbonic anhydrase III in 100 mM sodium phosphate buffer of pH 6.90. This unusually high value, reflecting the high tryptophan content, was obtained both by calibration against biuret measurements and by calculations according to the method of Wetlaufer (45). No significant differences were noticed for the extinction coefficients of the monomer and the dimer. Simi- larly, the ratio of the absorbance at 280 nm to that at 260 nm was 1.8 for both species.

Zinc Colztelzt-Based upon zinc analyses of three different carbonic anhydrase preparations the stoichiometry of zinc atoms per muscle carbonic anhydrase monomer (M, = 29,000) was determined to be 1.02. The direct overlap of the zinc profile with that of the protein in the final purification (Fig. 2) is an indication of both the constant ratio of zinc to protein and the homogeneity of the material obtained in the chromatographic peak. As mentioned previously (17), the COZ hydratase activity is directly proportional to the amount of zinc present in the enzyme molecule and successive removal of zinc is accompanied by a parallel decrease in the specific activity.’

Amino Acid Composition-Numerous amino acid analyses were performed under controlled conditions as previously described (25). The results are summarized in Table II, in which the average values for all rabbit muscle carbonic anhydrase III samples are compared with values obtained for

carbonic anhydrases I and II isolated from rabbit erythrocytes (19). To allow direct comparison, all values are normalized to a molecular weight of 29,000 for the monomer.

The table shows convincingly that, although there are overall similarities among the three carbonic anhydrase species, all three are clearly different. The similarities extend to (h) a high proportion of basic and acidic amino acids, (b) a relatively

TARIX I

Summary of general properties of rabbit muscle carbonic anhydrase III

Monomer I>imer

SLL 3.2 4.7 Molecular weight

Sedimentation equi- 29,000 + 500 56,000 -I- 900 librium”

Gel filtration” 27,000 f 4,000 52,000 f 9,000 Sodium dodecyl sul- 28,000 f 500

fate gel electrophoresis’

Best value 29,000 58,000”

Isoelectric pH’ 8.41 + 0.12 9.34 +- 0.06 0 ,‘A &HO 2.32 2.32

Zinc content (g atoms 1.02 2.04 of zinc/m01 of protein)’

” For a range of protein concentrations from 0.1 to 5 mg/ml. ’ Mean of four determinations each. ’ Mean of eight quadruplicate determinations from Koester et al.

(17). ” Calculated as 2 x 29,000. ’ Mean of five separate determinations. ’ Based on M,. of 29,000 and 58,000, respectively, from Koester et al.

(17).

TARI.E II

Comparison of amino acid compositions of carbonic anhydrases from rabbit muscle (CA III) and rabbit erythrocytes (CA I and

CA II)

Residues calculated for an M, of 29,000” Amino acid residue

CA Ih CA II” CA III’

Lysine 20.1 22.0 17.8 Histidine 11.4 14.6 10.7 Arginine 3.9 9.3 12.6 Aspartic Acid 32.0 27.4 27.9 Threonine 10.0 13.7 11.2 Serine 30.6 16.6 19.3 Glutamic Acid 20.9 23.7 18.8 Proline 17.1 19.1 22.5 Glycine 16.9 19.9 18.8 Alanine 21.1 10.7 14.4 Valine 16.9 15.3 14.1 Methionine o-1 2-3 1.9 Isoleucine 8.5 10.5 10.3 Leucine 25.0 22.8 19.6 Tyrosine 8.5 7.4 8.6 Phenylalanine 11.8 12.4 11.5 Tryptophan” 6.6 7.1 9.7 Cysteine’ 1 2 5.8

n For comparative purposes, the residue numbers are based on the same molecular weight of 29,000 for all three species.

’ Values obtained from Walther et al. (19) and recalculated for a molecular weight of 29,000. These values represent recoveries after 20 h of hydrolysis in 6 N HCl, and may, therefore, not be directly comparable to CA III for certain residues.

’ Values represent an average of 12 to 20 analyses of individually hydrolyzed protein samples, with an overall precision of +2%. Maxi- mal recoveries and extrapolations to zero time hydrolysis were calculated, where required, from a separate hydrolys’is time study.

‘Determined according to the method of Edelhoch (26). ’ Determined for the monomer species as cysteic acid after per-

formic acid oxidation (27), and by titration of free -SH groups with p-mercuribenzoate (28).

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4148 Carbonic Anhydrase III of Rabbit Muscle

high proline content, (c) a low methionine content, and (d) the presence of a significant number of aromatic amino acid residues. Carbonic anhydrase III appears to have an unusually high content of tryptophan and tyrosine and to be especially rich in arginine. The most distinctive feature of carbonic anhydrase III, however, and its greatest difference in composition as compared with carbonic anhydrases I or II, is the presence of 6 cysteine residues. No more then 2 cysteine residues have previously been found in any mammalian carbonic anhydrase (3), although more have been seen in lower animal and plant forms as referred to in more detail under “Discussion.”

Sulfhydryl and Total Half-cystine Content-The pro- nounced effect of dithiothreitol on the monomer-dimer distribution pattern on Sephadex G-75 suggested that oxidation of sulfhydryl groups might be involved, including the possibility that the dimer is the result of intermolecular disulfide formation in the absence of a sulfhydryl reducing agent. A detailed study was, therefore, undertaken involving sulfhydryl analyses byp-mercuribenzoate titration and measurements of the total half-cystine content by performic acid oxidation of numerous monomer and dimer samples, the results of which are shown in Table III. All of the -SH groups of the monomer species were moderately accessible (within 30 min) to p-mercuribenzoate without the addition of denaturant. The data show clearly that both species have the same contents with respect to total cysteic acid (approximately 6 residues per monomer equivalent) but that the dimer has 1 less sulfhydryl residue per monomer equivalent (or 2 less per dimer molecule). These data are in good agreement with our proposal that the dimer is produced by formation of a single disulfide bond between two identical monomers.

Peptide Mapping-Peptide maps prepared from tryptic digests of guanidine hydrochloride-denatured and iodo- [‘*C]acetate-carboxymethylated carbonic anhydrases I, II, and III are shown in Fig. 7. Each map is a composite drawn from 15 or 16 individual maps of each respective isoenzyme for

which the slight differences in mobility have been corrected to refer to common coordinates. On the basis of their lysine and arginine contents, 25 peptides are expected for carbonic anhydrase I and 32 peptides each for II and III. We have been able to identify 25 peptides for carbonic anhydrase I and 30 each for II and III, which is in good agreement with the data from amino acid analysis. All three isoenzymes have one peptide spot (No. 3) corresponding to free arginine, and carbonic anhydrases I and II have, in addition, one that co- migrates with free lysine (No. I). Peptides from each isoenzyme that are possibly homologous to each other are listed in

TABLE III

Cysteine and total half-cystine contents of rabbit muscle carbonic anhydrase III

Residues/molecule” Method

M0n0mer 111iiler

p-Mercuribenzoate” 5.87 +- 0.12 (17) 9.88 + 0.26 (13) (cysteine)

Performic acid oxi- 5.73 * 0.04 (5) 11.83 + 0.09 (3) dation’ (half-cystine)

” Calculated for molecular weights of 29,000 for the monomer and 58,000 (i.e. 2 X 29,000) for the dimer, respectively. An Ei&;,,, of 2.32 was assumed for both species. Numbers in parentheses indicate the number of determinations.

’ Sulfhydryl content measured by Boyer’sp-mercuribenzoate assay (28) in the presence of sodium dodecyl sulfate.

’ Cysteic acid content after performic acid oxidation (27), corrected for 100% recovery by calibration with performic acid-oxidized gluta- thionine (recoveries ranged from 92.5 to 94%).

CA I

r:;ORlGlN

CA II

5 ORIGIN

CA III

0 b ELECTROPHORESIS (2)'

FIG. 7. Comparison of tryptic peptide maps of carbonic anhydrases (CA) I and II from rabbit erythrocytes and carbonic anhydrase (CA) III from rabbit skeletal muscle. For experimental conditions, refer to “Methods.” Indication by special stains is as follows: vertical lines, phenanthrenequinone stain for arginine; dots, Ehrlich’s reagent for tryptophan (also visible without spraying by natural fluorescence under UV light); horizontal lines, cysteine peptides identified by carboxymethylation with iodo[‘%]acetate. All spots were also visualized with fluorescamine, except for CA I-14 (arginine only) and CA II- 12 and CA III- 12 (tryptophan only).

Table IV. These results indicate convincingly that, although the three proteins are clearly distinct from each other, carbonic anhydrase III is more similar to II than it is to I. Of special interest are the homologous peptides CA II-12 and CA 111-12, which stain for tryptophan but not with fluorescamine, indicating that their NHa-terminal group is not free. Carbonic anhydrase NHa-terminal amino acids are generally acetylated (3-5), and Ferrell et al. (46) have recently shown that rabbit blood carbonic anhydrase II has an acetylated NHZ-terminal lysine peptide containing tryptophan, which may be equiva-

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TAHIX IV

Some probable peptide homologies between carbonic anhydrases Z, ZI, and III

CA1 CA II CA III

Tryptic maps without carbamylation

1” 1” 2 2 3h 3h 3”

4 4 6 6 7 7 8 8 8

9 9 11 11 12 12 21 21 27 27

Tryptic maps with carbamylation

lb 1” 2 2

3 3 4 4 5 5

10 10

‘I Spot co-chromatographs with free lysine. ‘Spot co-chromatographs with free arginine.

lent to our peptide CA 11-12. The rabbit muscle carbonic anhydrase III NH2-terminal peptide may, therefore, be acetylated as well, and possibly identical with the blood carbonic anhydrase II peptide.

Carbonic anhydrase III is primarily distinguishable from the other isoenzymes by its high cysteine content. However, only three spots were found to be labeled by iodo[‘%]acetate, suggesting that there may be more than 1 cysteine residue per such peptide or that not all 6 cysteine residues were equally amenable to carboxymethylation under the conditions chosen for alkylation. None of these spots correspond to the single cysteine-containing peptide of carbonic anhydrase I (No. 19) or carbonic anhydrase II (No. 14) which are known in the human isoenzymes I and II to occupy different positions in the primary sequence (47-49).

Maps prepared in analogous fashion from tryptic peptides of samples in which the lysine residues had been carbamylated so that cleavage would occur only at the arginine residues are shown in Fig. 8. Although both similarities and differences in these larger peptides tend to be somewhat obscured, there nonetheless appear to be several homologies (Table IV). Again, carbonic anhydrase III seems to be more similar to II than to I, supporting the conclusions drawn from comparative amino acid analyses (see above). It must be emphasized, however, that these results can only be regarded as tentative evidence and that the actual extent of homology between carbonic anhydrase III and the other two isoenzymes must await elucidation of their complete amino acid sequences.

DISCU.S.SION

Nomenclature-Data pertaining to its amino acid composition and immunochemical properties presented under “Re- sults” and in a previous communication (17) provide evidence that Basic Muscle Protein is a carbonic anhydrase whose structure is sufficiently different from that of either carbonic anhydrase I or II to suggest that it is coded for by a gene different from either. Following the proposal of Tashian and Carter (4), according to which the roman numerals “I” and “II” are to be used for the genetically distinct low and high activity forms, respectively, of blood carbonic anhydrase and “III,” “IV,” etc. are to designate additional, genetically distinct

t

CA I

5: ORIGIN

CA II w $7

:2 ORIGIN

CA III

:3 ORIGIN

0 * ELECTROPHORESIS (2)

FIG. 8. Comparison of tryptic peptide maps of carbamylated carbonic anhydrases (CA) I and II from rabbit erythrocytes and of carbamylated carbonic anhydrase III from rabbit muscle. Experimen- tal conditions were as described under “Methods,” except that non- radioactive iodoacetate was used for the carboxymethylation of cysteine. BZack spots represent peptides visualized with fluorescamine; dotted spots, peptides with natural fluorescence under UV light.

carbonic anhydrase species as they aye discovered, the appropriate designation for the muscle isoenzyme is, therefore, carbonic anhydrase III.

General Similarities to and Differences from Other Car-

bonic Anhydrases-In terms of its gross features (molecular weight, sedimentation coefficient, zinc content, and depend- ence on zinc as a cofactor for activity), carbonic anhydrase III is a typical carbonic anhydrase like carbonic anhydrases I or II. At the level of primary structure, although still related to its isoenzymes, it is a clearly distinct species. Specifically, it

differs more from carbonic anhydrase I than from II, if one compares both the total amino acid contents and the results of comparative peptide mappings. Both carbonic anhydrases

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4150 Carbonic Anhydrase III of Rabbit Muscle

II and III have substantially more basic amino acids than I. In addition, carbonic anhydrase I has a relatively higher serine content than II or III, a pattern which has been a general criterion of distinction between low activity type I isoenzymes and high activity type II forms (3). Tashian and co-workers (6), on the basis of recently implemented peptide mapping and sequence studies of low activity bovine carbonic anhydrases, have also come to the conclusion that bovine carbonic anhydrase III is sufficiently different from the corresponding I and II isoenzymes to be considered a genetically different species, while substantial sequence homologies (particularly in the active site region) between carbonic anhydrase III and the other two bovine isoenzymes clearly show it to be a true carbonic anhydrase. Like the rabbit muscle enzyme, bovine muscle carbonic anhydrase III is lower in activity than either isoenzyme I or II, has poor esterase activity, and is relatively sulfonamide-resistant’ (6).

Our present data and those previously reported (17) are at variance with those of Holmes (16) for sheep carbonic anhydrase species for which he has described antibodies to CA-A (carbonic anhydrase III, presumably) that cross-react with CA-B (carbonic anhydrase I) in a zymogram of muscle tissue extract, a claim which would imply that sheep muscle contains a type I carbonic anhydrase in addition to what appears to be the muscle type III form. We believe it is misleading to identify any carbonic anhydrase activity bands purely on the basis of their electrophoretic mobility as either I, II, or III, if such bands are not also characterized both in terms of their relative resistance to inhibition by sulfonamides and their specific activity to establish whether they are the high or low activity type. Our findings, as well as those of Deutsch et al. (44), show clearly that simple changes in the sulfhydryl oxidation state alone are sufficient to produce differences in the net charge of protein molecules resulting in enzyme species with different electrophoretic mobilities. It is notable that Scopes (12) had also observed a minor electrophoretic band of his Protein F that appeared during storage and increased in intensity with time of storage. In light of these observations, it appears more likely that Holmes’ multiple muscle carbonic anhydrase activity bands are sulfhydryl oxidation artifacts, or other carbonic anhydrase III-type variants, than that they are genuine carbonic anhydrase I or II isoenzymes.

Sulfhydryl Content-The most distinct difference in amino acid composition between carbonic anhydrase III and other mammalian carbonic anhydrases is its cysteine content, with rabbit muscle carbonic anhydrase III having 6 cysteine residues and the others from 0 to 2 (3). The total absence of cysteine from some mammalian isoenzymes suggests that it is not essential for catalysis, although the plant enzymes are inhibited by p-mercuribenzoate (14, 50, 51). Nonmammalian carbonic anhydrases with high cysteine contents are from widely differing species, and they are all (in contrast to carbonic anhydrase III) functionally classified as high activity forms. In addition, all, except for the plant enzymes, are sulfonamide-sensitive. Of the plant carbonic anhydrases, the parsley enzyme has 7 cysteine residues/subunit of 29,000 molecular weight (22). Among animal species, two genera of shark have been found to contain carbonic anhydrases with 18 and 25 half-cystine residues, respectively, which are involved in internal disulfide linkages (52); frog carbonic anhydrase has 4 cysteine residues which are accessible to titration by p-mercuribenzoate (53); and for chicken erythrocyte carbonic anhydrase, a cysteine content of 7 residues has been reported (54).

Monomer-Dimer Interconversion-One of the outstanding features of carbonic anhydrase III is its existence as two

’ R. E. Tashian, personal communication, July, 1977.

clearly separable forms of different molecular size. This property provided considerable puzzlement in the beginning and contributed to the delay in identifying Basic Muscle Protein as a carbonic anhydrase since the existence of a monomer- dimer equilibrium had not been reported for the more exten- sively characterized carbonic anhydrase species. We believe that the data presented in this paper provide the first conclu- sive evidence for the existence in mammals of two individually identifiable and physically separable carbonic anhydrase forms that have a monomer-dimer relationship to each other, whose molecular weights have been determined by rigorous ultracentrifugal methodology, and for which the dimer has been shown by differential amino acid analysis to be the disulfide oxidation product of the sulfhydryl monomer.

Qualitative evidence for dimer formation has also been reported for chicken erythrocyte carbonic anhydrase (54). For the latter, the monomer was found to be a high activity carbonic anhydrase with respect to both CO2 hydration and esterase activity but the dimer to be much less active. More- over, the chicken enzyme was found to require sulfhydryl reducing agents for full expression of its activity. In contrast, rabbit muscle carbonic anhydrase III is equally active as a carbonic anhydrase whether it is in the dimer or the monomer form.” The rabbit muscle dimer is similar to the avian erythrocyte dimer, however, in that both represent the minor species, i.e. without the reducing agent present, the dimer amounts usually to no more than 20% of the total carbonic anhydrase protein. Even under the deliberately induced, relatively rigorous oxidizing conditions described under “Re- sults” for the rabbit muscle enzyme, it has never been possible to convert the monomer entirely to the dimer form. Plant carbonic anhydrases, on the other hand, have different properties in that their native molecular configuration is that of hexamers which require both guanidine hydrochloride and sulfhydryl reducing agents to produce the monomer species (14, 50).

In addition to these instances in which carbonic anhydrases with relatively high cysteine content have been shown to exist as dimers or multimers, another interesting case of dimeriza- tion has recently been reported for a carbonic anhydrase of low sulfhydryl content. Deutsch et al. (44) have described a point mutation of horse erythrocyte carbonic anhydrase II in which a cysteine residue is substituted for arginine 180. This cysteine differs from the one other normally present by being highly reactive and forming primarily a glutathione derivative as well as resulting in the formation of small amounts (less than 10% of the total) of dimer.

Functional Significance-The physiological role of the lower activity carbonic anhydrase is currently a matter of intense study in several laboratories. Since the high activity erythrocyte carbonic anhydrase II has a catalytic capacity of CO, hydration exceeding that required for CO2 exchange through the lungs, Maren et al. (55), for example, have proposed that the function of the low activity erythrocyte isoenzyme I is not that of COz hydration. Since muscle carbonic anhydrase III has an even lower CO:! turnover than isoenzyme I, it might be argued that its function should also not be that of a CO2 hydratase, particularly since muscle has traditionally been considered to be a tissue for which carbonic anhydrase is “an enemy of the organism” (56). Yet carbonic anhydrase III has no activity at all as a ,&naphthylacetate esterase (17). This is in contrast to the type I enzyme for which high esterase capacity relative to the type II enzyme is considered a char- acteristic feature (14). Overall, carbonic anhydrase III has much less catalytic versatility than either of the erythrocyte isoenzymes” (17), so that a physiological role for it as an

(’ M. K. Koester and E. A. Noltmann, unpublished experiments.

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esterase is highly improbable. One needs, therefore, to consider other functional alterna-

tives that may result from its catalyzed reaction that involves the substrates H+, HCOZm, COZ, and HzO. The levels of any or all of these in muscle could conceivably be regulated by carbonic anhydrase III. The maintenance of this catalytic equilibrium, for instance, has the effect of converting meta- bolically generated CO2 (which readily permeates the cell membrane) to HCOam (to which the cell membrane is relatively impermeable) (57) which, in turn, will influence the rate at which COZ can leave the cell. In that regard, it is interesting to note that facilitated COZ diffusion has been demonstrated in vitro (58, 59) and its observation in muscle has been interpreted to imply the existence of carbonic anhydrase in that tissue (60).

The effect of carbonic anhydrase. III on maintaining the CO,/HCO,~ equilibrium would be expected to be even more important under conditions of metabolic stress, i.e. the presence of excess CO2 (hypercapnia) or excess H’ (acidosis). In fact, both cardiac and skeletal muscle have been found to have considerable capacity for buffering excess CO2 (61), perhaps as large as that of blood (62), whereas evidence regarding the capacity of muscle to buffer H’ ions is conflicting (62-64). These results were obtained with different muscle types and under different conditions4 and may require rein- vestigation in light of recent findings by Holmes (66) that red (“slow,” preferentially aerobic) muscle contains considerably more carbonic anhydrase than white (“fast,” preferentially anaerobic) muscle and by Aickin and Thomas (65) that slow muscle fibers have 3 times the CO2 buffering capacity of fast muscle fibers. The latter authors have also found that the membrane potential of red muscle is more sensitive to varying CO, levels than that of white muscle, indicating a greater need for COa regulation in red muscle. Thus, there is increasing evidence for a possible link of carbonic anhydrase to the regulation of CO, concentration and of pH in muscle. We therefore consider it a most reasonable hypothesis to propose that involvement in this regulation is the physiological function of muscle carbonic anhydrase. This hypothesis, however, must remain tentative until in uiuo studies have been performed which take into account the unique properties of this enzyme.”

Acknowledgments-We wish to thank Dr. Richard Tashian for generously providing us with advance information on sequence data for the low activity bovine carbonic anhydrases, Dr. Ning Pon for assistance with the ultracentrifuge experiments, and Dr. Gary Hath- away for some of the preliminary ultracentrifuge studies and for initially noting the similarity in amino acid composition of Basic Muscle Protein to some of the carbonic anhydrases.

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A M Register, M K Koester and E A Noltmannidentity with "basic muscle protein".

Discovery of carbonic anhydrase in rabbit skeletal muscle and evidence for its

1978, 253:4143-4152.J. Biol. Chem.

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