PURIFICATION OF MYELIN CARBONIC ANHYDRASE VICTOR
s. SAPlRSTEIN and
MARJORIE B. LEES
Department of Biochemistry, The Eunice Kennedy Shriver Center, Waltham, MA 02154 and Department of Biological Chemistry. Harvard Medical School, Boston, MA 021 15, U.S.A (Rereiced 28 November 1977. Accepted 21 February 1978)
Abstract-A procedure has been developed for the purification of the membrane bound form of carbonic anhydrase from rat brain myelin. The procedurc is rapid, requiring only two steps, and can be applied to small amounts of material. Conditions have been established whereby the enzyme can be almost quantitatively solubilized with up to a 60 fold increase in specific activity. Purification by afinity chromatography yields a preparation which is homogeneous by polyacrylamide gel electrophoresis. However, preliminary evidence suggests that activity may be reduced by the removal of lipids during chromatography and subsequent dialysis. The purified preparation is high in dicarboxylic and hydroxyl amino acids and contains only 1-2 cysteine residues. The reduction of cysteine appears to be essential for the full expression of enzymatic activity.
CARBONIC anhydrase (EC 4.2.1.1) is a widely distributed enzyme existing in both a soluble and membrane bound form (MAREN,1967; KARLER& WOODBURY, 1960). In most tissues the soluble enzyme predominates and this form has been studied extensively (see MAREN,1967; CARTER, 1974). Several soluble isozymes exist and these differ in their tissue distribution, amino acid composition and sensitivity to inhibitors. Little is known of the membrane bound form of the enzyme. In brain, more than half of the carbonic anhydrase is membrane bound and several groups have reported that a portion of the membrane bound enzyme occurs in purified myelin (CAMMER et a/., 1976; YANDRASlTZ et a/.,1976; SAPIRSTEIN & LEES, 1977). The association of carbonic anhydrase with acid-base balance and ion and fluid fluxes in many tissues including brain and choroid plexus (MAREN, 1967; DAVSONKL SEGAL, 1970: BOURKEet al., 1975) suggests that the enzyme may participate in similar functions in the myelin sheath. To understand the function and properties of the myelin enzyme and to compare it with the soluble enzyme, a purified preparation is required. The present paper reports on a simple procedure for the purification of myelin carbonic anhydrase. MATERIALS A N D METHODS Materials. All chemicals were reagent grade. Human and bovine red blood cell carbonic anhydrase and acetazolamide were obtained from Sigma Chemical Co., St. Louis, MO. p-Aminomethylbenzenesulfonamide (PAMBS) was purchased from Aldrich Chemical Co., Milwaukee, WI, Sepharose CL-4B from Pharmacia Fine Chemicals. Piscataway, NJ and Triton X-100from Fisher Chem. Co., Pittsburgh, PA. Sprague-Dawley rats (18G250 g) were Abbreciatiorzs used: SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.
obtained from Charles River Breeding Laboratory, WilmIngton, MA. Carbonic anhydrase assay. Carbonic anhydrase activity was measured using a micromodification of the Maren procedure (MAREN1960). Briefly, 10-50 pl aliquots of sample were added to 500p1 of a phenol red solution (1.25 mgS/,) containing 2.6 mM-Na2C0, saturated with CO,. After the addition of 70 p1 of bicarbonate buffer, pH 9.0 (0.3 ~-Na,CO,-0.206 M-NaHCO,), the time required for the indicator color to shift from red to yellow was recorded. The uncatalyzed time was determined by carrying out the assay in the absence of enzyme. The units of activity are expressed as .~ - catalyzed time uncatalyzed time catalyzed time
One unit of activiiy therefore corresponds to a 50% reduction from the uncatalyzed time. Isolation of myelin. Myelin was isolated from brains of Sprague-Dawley rats as described by NORTON & PODUSLO (1973). The material obtained after the second gradient separation was washed 3 times with distilled water to remove all of the sucrose. To determine possible binding of soluble components to the myelin. preparations were assayed spectrophotometrically for lactic dehydrogenase (LDH) (BERGMEYER, 1967). No L D H activity was detected; under rhe assay conditions used the presence of as little as 0.XYi LDH would have been detectcd. The myelin was either assayed for carbonic anhydrase activity immediately or kept frozen at -20". N o loss of activity was observed after storage of the frozen myelin for 2 weeks. Triron-DTT extraction of niyelin. Myelin was extracted at room temperature by periodic homogenization in a Dounce homogenizer with various concentrations of Triton containing 0.1:~; (wiv) DTT. After 20min, the preparation was centrifuged at 30,000 g for 15 min. Release of carbonic anhydrase activity was determined by assay of both the supernatant and the residue. Preparation of ajfiniry column. p-Aminomethylbenzene sulfonamide (PAMBS) was coupled to cyanogen bromideactivated Sepharose CL-4B by the procedure of WHITNEY (1974). The extent of coupling of ligand corresponded t o
505
506
ind MARJORIE B. LEES VICTORS. SAPIRSTEIN
2 5 p n o l of inhibitor per gram of gel. After reaction with PAMBS, the remaining reactive sites were blocked with 0.5 M-monoethanolamine buffered with 0.5 M-NaHCO, to pH 9.4. The reaction was terminated by washing the gel with a large volume of IOmM-NaHCO, and the gel was stored at 4" in 10 mM-NaHCO, containing 0.027: sodium azide. Prior to use, the gel was washed free of salts and equilibrated with Triton-DTT. Unless otherwise stated, the term Triton-DTT refers to 0.5?', Triton X-100 containing 0.1% dithiothreitol. A,fiYi,tiry chromatography. A 1.0 x 3.0 cm iolumn was prepared. The amount of gel (2.&2.5ml) in the column is theoretically sufficient for the binding of up to 2 0 m g of enzyme (WHITNEY, 1974). In actual practice samples containing significantly less than one mg of carbonic anhydrase were applied. The Triton-DTT extract of myelin was passed through the PAMBS-Sepharose affinity column. The freely eluting material and a subsequent two'.column volume wash with Triton-DTT were pooled and assayed for carbonic anhydrase and protein. The column was then washed with 6 vol of 25 mM-NaCI in Triton-DTT diluted 1 : 10 with 25 mM-Tris-sulfate pH 8.0. After dialysis against water, the eluate was assayed in the presence of 0.1% DTT. The enzyme was eluted from the column by washing with 6 column volumes of 25 mM-NaC1 containing 25 mM-Trissulfate p H 8.0 and M-acetazolamide. The eluate was dialyzed in turn against water, 0.5 M-NaCI adjusted to pH 6.5 with sodium acetate, and then water. The retentate was lyophilized and stored in a desiccator at - - 2 O . SDS-polyacrylarnide gel electrophoresis. The lyophilized samples were extracted with acetone t o remove residual Triton X-100 and were dissolved in a solution containing 1% SDS, 1% DTT and 8% sucrose. SDS-polyacrylamide gel electrophoresis was carried out on 10% gels by the (1969) after pre-electrophormethod of WEBFR& OSBORNE esis of the gels for 30 min at 5 mA/tube to remove residual persulfate (ZAHLER, 1974). The gels were stained with Coomassie Brilliant Blue for 8 h and destained in methanolwater-acetic acid 1 : 1:0.22. Analytical procedures. Proteins were determined by the method of LOWRY e f a!. (1950) as modified by LEES & PAXMAN (1974). Amino acids were determined on a Beckman Model 119C analyzer after hydrolysis in 6 N-HCl in mcuo for 24 h.
RESULTS Carbonic anhydrase actiuity: effects of Triton and D T T
The carbonic anhydrase activity of myelin suspended in either water or 0.5% Triton was 4.1 units TABLE1. CONDITIONS FOR
Water 0.5% Triton X-100 Water + DTT 0.5"/, Triton X-100
Extraction of myelin carbonic anhydrase
The extraction of myelin with Triton-DTT is dependent on the concentration of the detergent (Fig. 1). At a concentration of 0.05% Triton, little or no enzyme was solubilized whereas a maximum amount of enzyme is extracted with 1% Triton. Higher concentrations of Triton appear to be inhibitory. The extraction of carbonic anhydrase was also dependent upon the volume of Triton-DTT used per mg of myelin protein. When 3 mg of myelin protein was suspended in l m l 0.5% Triton-DTT, the membrane bound enzyme was solubilized quantitatively by three serial extractions (Fig. 2). Negligible activity remained in the residue. However, if the ratio of protein to volume of Triton-DTT was doubled, more than a 3-fold drop was observed in the efficiency of the extraction. For the purpose of purification of the enzyme, the following solubilization procedure was found to be most efficient: myelin was extracted with 0.05% Triton-DTT followed by a single extraction with 0.5% Triton-DTT. The first extraction removed non-enzymatic protein with no loss of enzyme. The subsequent extraction with 0.5% Triton-DTT solubilized more than 75% of the enzyme and resulted in close to a 50-fold increase in specific activity to a value of 544
ASSAY AND SOLUBILIZATION OF MYELIN CARBONIC: ANHYDRASE
Total units*
Assay condition a. b. c. d.
per mg protein (Table 1). When myelin was suspended in DTT prior to assay, the activity was increased 3-fold regardless of the presence or absence of Triton. Almost half of the enzyme activity was solubilized by a single extraction with Triton-DTT and advantage was taken of this observation for the purification of the myelin carbonic anhydrase (described below). O n the other hand, only a small portion of the enzyme was solubilized by Triton alone. If the Tritonsoluble fraction is incubated in DTT prior to assay, a 3-fold increase in activity is again observed. The activity of the Triton-soluble fraction, even when assayed in the presence of DTT, is significantly less than the activity of the Triton-DTT soluble fraction. These observations suggest that DTT facilitates the dissociation of the enzyme from the- membrane but that there is an additional effect of DTT on the activity which is independent of its effect on solubilization.
+ DTT
* From 14.6 mg myelin protein. t Myelin was extracted once at a
Units/mg protein
60
4.1
60 182 182
4.1 12.5 12.5
Total units solubilizedt by Triton Triton + DTT
16 50
85
protein concentration of 3.3 mg/ml with either Triton or Triton f DTT. The material solubilized with Triton alone was assayed either without (b) or with (d) added DTT
Myelin carbonic anhydrase
70 60
507
-
25
FIG.2. Serial extraction of myelin with 0.50,; Triton X-100 + 0.1% DTT. Myelin was extracted as described in Fig. 1 and in Methods. The myelin residuc was resuspended in Triton X-100 + DTT prior to assay.
TRITON CONCENTRATION (vol. %)
Frc. 1. Extraction of myelin carbonic anhydrase with increasing amounts of Triton X-100. Myelin (3.3mg protein/ml) was extracted at room temperature for 20 min by periodic homogenization in a Dounce homogenizer in the presence of 0.1% dithiothreitol. Solubilization was assessed by assay of the supernatant after centrifugation at 30,000 g for 15 min.
washing step removes non-specifically bound protein and maintains the column at pH 8.0 which is required for maximum binding of the eluting inhibitor in the following step. Subsequent elution of the column with M-acetazolamide reproducibly results in an enzyme preparation with a specific activity between 900 and 2000 units/mg protein corresponding to over a 100-fold purification. The homogeneity of the myelin carbonic anhydrase preparation is suggested by the single band observed on SDS gels when relatively large amounts of protein are used (Fig. 4). The molecular weight of the purified myelin carbonic anhydrase corresponds to 30,000 daltons since it migrates in SDS gels to the same position as the soluble red blood cell enzyme. Upon lyophilization, the purified enzyme loses both activity and solubility. As a consequence, electrophoresis in non SDS gels has not been possible. This is in contrast to myelin which can be lyophilized without loss of activity (CAMMER et al., 1976; Sapirstein & Lees, unpublished). The amino acid composition is characterized by relatively large amounts of aspartic acid, glutamic acid, serine, alanine and glycine (Table 3). Dicarboxylic acids account for 19.6% of the total amino acids recovered whereas basic amino acids account for only 12.9%. The cysteine content, based on recovery of cysteic acid is between one and two residues per mol. The protein is particularly low in S-containing and in aromatic amino acids.
units per mg protein (Table 2). Carbonic anhydrase has been enriched in this fraction to such an extent that a band corresponding to soluble carbonic anhydrase is clearly visible on gels of the 0.5% Triton-DTT extract whereas it is not detected on gels of the original myelin (Fig. 3). AfJinity chromatography
Preliminary studies showed that red blood cell carbonic anhydrase can be quantitatively adsorbed to PAMBS-Sepharose CL-4B in the presence of Triton and can be dissociated from the matrix with high concentrations of a soluble inhibitor. Advantage was taken of this observation to purify the solubilized carbonic anhydrase. The Triton-DTT solubilized fraction of the myelin is chromatographed on a PAMBSSepharose column pre-equilibrated with Triton-DTT. Carbonic anhydrase activity is retained on the column and is not eluted with either Triton-DTT or Triton-DTT diluted 1: 10 and containing 50 mu-Tris sulfate pH 8.0, (Table 2). The gel pattern of the column eluate reflects the selective deletion of the carbonic anhydrase band (Fig. 3-gel 3). The Tris sulfate TABLE2. PURlFlCATloN OF
MYELIN CARBONIC ANHYDRASE
Total units
Myelin 0.05% Triton-DTT extract 0.5% Triton-DTT extract Sulfonamide affinity Column* (1) Triton-DTT eluate (2) Triton-DTT + acetazolamide eluate
Protein (mg)
Specific activity
1050
98 1.90 1.93
13.7 44 544
39 500
1.80 0.24
21.7 2083
1340 84
Yield
% -
6.3 78
2.9 31.3
* PAMBS-Sepharosecolumn eluted with (1) 0.5% Triton-0.17; DTT and then washed with NaCl-Tris sulfate buffer in Triton-DTT as described under M-acetazolamide. Methods and (2) same but containing
VICTORS. SAPIRSTEIN and MARJORIE B. LEES
508 TABLE3. AMINOA C I D
COMPOSITION OF MYELIN CARBONIC ANHYDRASE
mol percent I 2
Amino acid Aspartic Threonine Serine Glutamic Proline Glycine
9.5 5.6 12.1 10.1 4.6 12.6 8.8 0.8 5.4
Alanine
112 Cystine* Valine
.o
Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
* Determined
1
4.0 7.3 2.0 3.7 3.4 6.3 3.2
9.3 4.6 11.6 10.8 4.9 13.1 8.5 ~
5.5 1.2 3.9 7.2 2.6 3.4 3.4 6.4 3.3
as cysteic acid.
DISCUSSION
This paper describes the first purification of a membrane bound carbonic anhydrase not only from brain but from any tissue. The procedure is simple and rapid and can be carried out on myelin obtained from a single rat brain. It should therefore be useful for metabolic studies on the regulation of myelin carbonic anhydrase. The hundred-fold increase in specific activity shown in Table 2 may, in fact, be an underestimation of the extent of purification. After affinity chromatography the specific activity increases only 4-fold over that of the solubilized preparation. However, the gel electrophoretic patterns of these fractions suggest greater purification. The specific activity of the purified enzyme may be low either because of incomplete removal of the inhibitor used to elute the columns or because of a requirement of the enzyme for lipid. At present there is insufficient data to distinguish between these possibilities. An important determinant of enzyme activity appears to be the maintenance of cysteine in the reduced form. Dithiothreitol has a significant effect on myelin carbonic anhydrase activity independent of whether the enzyme is membrane bound or solubilized. Therefore DTT seems to have a direct effect on the enzyme which is not mediated via effects on the intact membrane structure. Carbonic anhydrase from other tissues contain a single cysteine and the myelin enzyme is similar with a cysteine content of one to two residues (1.5 residues based on a molecular weight of 30,000). The marked increase in activity expressed after reduction with DTT suggests that the oxidation-reduction of cysteine could provide a physiologic control mechanism. The amino acid composition is characterized by relatively large amounts of dicarboxylic amino acids, glycine. alanine and serinc. To the best of our knowl-
edge the composition of rat brain soluble carbonic anhydrase has not been reported but the values we obtained are similar to those found in soluble carbonic anhydrases from other species (CARTER1972). Carbonic anhydrase can exist as at least two different isozymes and the high serine content is typical of the B isozyme. However, the myelin enzyme is unaffected by imidazole (SAPIRSTEIN& FLYNN,in preparation), a specific inhibitor of the B isozyme (KANNANet al., 1977). On the other hand, the solubilized myelin enzyme is refractory to chloride (SAPIRSTEINet al., 1977), a property which is typical of the C isozyme. Thus, the myelin enzyme appears to correspond to a C type isozyme. It is therefore similar to the soluble brain carbonic anhydrase which has been characterized in humans on the basis of its immunologic cross reactivity with the C isozyme from erythrocytes (FUNAKOSHI & DELJTSCH 1971). The high serine content observed in our preparations may therefore be a consequence of the presence of lipid serine. MCKINLEY & WHITNEY(1976) have suggested that the membrane bound carbonic anhydrase from kidney may be a distinct isozyme. A comparison of the soluble and myelin forms of the enzyme from brain requires purification of the respective proteins and we are currently purifying the soluble enzyme from brain. Ackrzowledge,nent--The study was supported in part by U.S. Public Health Service Grant NS 13649 and HD 05 155.
REFERENCES BERCMEYER H. U., BERNTE. & NESS B. (1965) in Methods of Enzymatic Analysis (BERGMEYER H. U., ed.). pp. 736-741. Academic Press, New York. BOURKER. S., KIMELBERG H. K., WESTC. R. & BREMMER A. M. (1975) J. Neurochem. 25, 323-328. CAMMER W., FREDMAN T., ROSE A. L. & NORTONW. T. (1976) J. Neurochem. 27, 165-171. CARTERM. J. (1972) Biol. Rev. 47, 465-513. DAVSON H. & SEGALM. B. (1970) J . Physiol. 209, 131-153. FUNAKOSHI S. & DEUTSCHH. F. (1971) J . biol. Chem. 246, 1088-1092. KANNANK. K., PETEFM., FRIDBERG K., CID-DRESDNER H . & HOVGREN S. (1977) FEBS Lelr. 73, 115-126. KARLER S. & WOODBURYD. M. (1960) Biochem. J . 75, 538-543. LEESM. B. & PAXWAN S. A. (1972) Anirlyt. Eiochem. 47, 184-1 92. LOWRY0. H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) J. biol. Chem. 193, 265-275. MARENT. H. (1960) J. Pharmac. exp. Ther. 130, 26-29. MARENT. H. (1967) Physiol. Rel;. 47, 595-781. MCKIVLEYD. N. & WHITNEYP. L. (1976) Biochim. biophy.7. Acra 445, 780-790. NORTONW. T. & PODUSLOS. (1973) J. Neurochem. 21, 749- 7 5 7. SAPIRSTEIN V. S. & LEES M. B. (1977) Trans. Am. Soc. Ncurncbcm. 8, 57 I . SAPIRSTFIN V. S.. TRACHTENHERG M. C., LEES M. B. & KOUL 0. in M \dinarion and Drn?yelinarion: Rccenr
509
FIG. 3. Purification of myelin carbonic anhydrase from one rat brain monitored by polyacrylamide gel electrophoresis. Gel 1, whole rat brain myelin, lOOpg protein; gel 2, material solubilized by Triton, 40pg protein; gel 3, material freely eluted from PAMBS Sepharose, S O p g protein; gel 4, material obtained after elution with acetazolamide: and gel 5, purified carbonic anhydrase standard, 25 pg protein. Electrophoresis was performed as described in Methods. All samples were added in a SO pI volume of 1%. SDS-O.I% DTT-4”/, sucrose. The anode is at the bottom of the figure. FIG.4. Comparison of polyacrylamide gels of purified myelin carbonic anhydrase and soluble carbonic anhydrase C. Gel 1, myelin carbonic anhydrase, 5 O p g protein and gel 2, soluble carbonic anhydrase C. 5 0 p g protein. Conditions as in Fig. 3.
Myelin carbonic anhydrase Chemictrl Adrarices (PALO J . & RIEKLNNEN P., eds.). Plenum Press. New York. In press. WEBER K. & OSBORNM. (1969) J. biol. Chem. 244, 4406-44 12. WHITNEY P. L. (1974) Ano/yr. Biorhem. 57, 467-476.
51 1
YANURASITZ J . R.. ERNSTS. A. & SALGANICOFF L. (1976) J. Neurochem. 27. 707-71 5. ZAiiLm W. L. (1971) In Merhods in Enzymoloqy Vol. XXXII. (FLEISCHER s. & PACKER L.,eds.) pp. 70-81. Academic Press. New York.
s. SAPlRSTEIN and
MARJORIE B. LEES
Department of Biochemistry, The Eunice Kennedy Shriver Center, Waltham, MA 02154 and Department of Biological Chemistry. Harvard Medical School, Boston, MA 021 15, U.S.A (Rereiced 28 November 1977. Accepted 21 February 1978)
Abstract-A procedure has been developed for the purification of the membrane bound form of carbonic anhydrase from rat brain myelin. The procedurc is rapid, requiring only two steps, and can be applied to small amounts of material. Conditions have been established whereby the enzyme can be almost quantitatively solubilized with up to a 60 fold increase in specific activity. Purification by afinity chromatography yields a preparation which is homogeneous by polyacrylamide gel electrophoresis. However, preliminary evidence suggests that activity may be reduced by the removal of lipids during chromatography and subsequent dialysis. The purified preparation is high in dicarboxylic and hydroxyl amino acids and contains only 1-2 cysteine residues. The reduction of cysteine appears to be essential for the full expression of enzymatic activity.
CARBONIC anhydrase (EC 4.2.1.1) is a widely distributed enzyme existing in both a soluble and membrane bound form (MAREN,1967; KARLER& WOODBURY, 1960). In most tissues the soluble enzyme predominates and this form has been studied extensively (see MAREN,1967; CARTER, 1974). Several soluble isozymes exist and these differ in their tissue distribution, amino acid composition and sensitivity to inhibitors. Little is known of the membrane bound form of the enzyme. In brain, more than half of the carbonic anhydrase is membrane bound and several groups have reported that a portion of the membrane bound enzyme occurs in purified myelin (CAMMER et a/., 1976; YANDRASlTZ et a/.,1976; SAPIRSTEIN & LEES, 1977). The association of carbonic anhydrase with acid-base balance and ion and fluid fluxes in many tissues including brain and choroid plexus (MAREN, 1967; DAVSONKL SEGAL, 1970: BOURKEet al., 1975) suggests that the enzyme may participate in similar functions in the myelin sheath. To understand the function and properties of the myelin enzyme and to compare it with the soluble enzyme, a purified preparation is required. The present paper reports on a simple procedure for the purification of myelin carbonic anhydrase. MATERIALS A N D METHODS Materials. All chemicals were reagent grade. Human and bovine red blood cell carbonic anhydrase and acetazolamide were obtained from Sigma Chemical Co., St. Louis, MO. p-Aminomethylbenzenesulfonamide (PAMBS) was purchased from Aldrich Chemical Co., Milwaukee, WI, Sepharose CL-4B from Pharmacia Fine Chemicals. Piscataway, NJ and Triton X-100from Fisher Chem. Co., Pittsburgh, PA. Sprague-Dawley rats (18G250 g) were Abbreciatiorzs used: SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.
obtained from Charles River Breeding Laboratory, WilmIngton, MA. Carbonic anhydrase assay. Carbonic anhydrase activity was measured using a micromodification of the Maren procedure (MAREN1960). Briefly, 10-50 pl aliquots of sample were added to 500p1 of a phenol red solution (1.25 mgS/,) containing 2.6 mM-Na2C0, saturated with CO,. After the addition of 70 p1 of bicarbonate buffer, pH 9.0 (0.3 ~-Na,CO,-0.206 M-NaHCO,), the time required for the indicator color to shift from red to yellow was recorded. The uncatalyzed time was determined by carrying out the assay in the absence of enzyme. The units of activity are expressed as .~ - catalyzed time uncatalyzed time catalyzed time
One unit of activiiy therefore corresponds to a 50% reduction from the uncatalyzed time. Isolation of myelin. Myelin was isolated from brains of Sprague-Dawley rats as described by NORTON & PODUSLO (1973). The material obtained after the second gradient separation was washed 3 times with distilled water to remove all of the sucrose. To determine possible binding of soluble components to the myelin. preparations were assayed spectrophotometrically for lactic dehydrogenase (LDH) (BERGMEYER, 1967). No L D H activity was detected; under rhe assay conditions used the presence of as little as 0.XYi LDH would have been detectcd. The myelin was either assayed for carbonic anhydrase activity immediately or kept frozen at -20". N o loss of activity was observed after storage of the frozen myelin for 2 weeks. Triron-DTT extraction of niyelin. Myelin was extracted at room temperature by periodic homogenization in a Dounce homogenizer with various concentrations of Triton containing 0.1:~; (wiv) DTT. After 20min, the preparation was centrifuged at 30,000 g for 15 min. Release of carbonic anhydrase activity was determined by assay of both the supernatant and the residue. Preparation of ajfiniry column. p-Aminomethylbenzene sulfonamide (PAMBS) was coupled to cyanogen bromideactivated Sepharose CL-4B by the procedure of WHITNEY (1974). The extent of coupling of ligand corresponded t o
505
506
ind MARJORIE B. LEES VICTORS. SAPIRSTEIN
2 5 p n o l of inhibitor per gram of gel. After reaction with PAMBS, the remaining reactive sites were blocked with 0.5 M-monoethanolamine buffered with 0.5 M-NaHCO, to pH 9.4. The reaction was terminated by washing the gel with a large volume of IOmM-NaHCO, and the gel was stored at 4" in 10 mM-NaHCO, containing 0.027: sodium azide. Prior to use, the gel was washed free of salts and equilibrated with Triton-DTT. Unless otherwise stated, the term Triton-DTT refers to 0.5?', Triton X-100 containing 0.1% dithiothreitol. A,fiYi,tiry chromatography. A 1.0 x 3.0 cm iolumn was prepared. The amount of gel (2.&2.5ml) in the column is theoretically sufficient for the binding of up to 2 0 m g of enzyme (WHITNEY, 1974). In actual practice samples containing significantly less than one mg of carbonic anhydrase were applied. The Triton-DTT extract of myelin was passed through the PAMBS-Sepharose affinity column. The freely eluting material and a subsequent two'.column volume wash with Triton-DTT were pooled and assayed for carbonic anhydrase and protein. The column was then washed with 6 vol of 25 mM-NaCI in Triton-DTT diluted 1 : 10 with 25 mM-Tris-sulfate pH 8.0. After dialysis against water, the eluate was assayed in the presence of 0.1% DTT. The enzyme was eluted from the column by washing with 6 column volumes of 25 mM-NaC1 containing 25 mM-Trissulfate p H 8.0 and M-acetazolamide. The eluate was dialyzed in turn against water, 0.5 M-NaCI adjusted to pH 6.5 with sodium acetate, and then water. The retentate was lyophilized and stored in a desiccator at - - 2 O . SDS-polyacrylarnide gel electrophoresis. The lyophilized samples were extracted with acetone t o remove residual Triton X-100 and were dissolved in a solution containing 1% SDS, 1% DTT and 8% sucrose. SDS-polyacrylamide gel electrophoresis was carried out on 10% gels by the (1969) after pre-electrophormethod of WEBFR& OSBORNE esis of the gels for 30 min at 5 mA/tube to remove residual persulfate (ZAHLER, 1974). The gels were stained with Coomassie Brilliant Blue for 8 h and destained in methanolwater-acetic acid 1 : 1:0.22. Analytical procedures. Proteins were determined by the method of LOWRY e f a!. (1950) as modified by LEES & PAXMAN (1974). Amino acids were determined on a Beckman Model 119C analyzer after hydrolysis in 6 N-HCl in mcuo for 24 h.
RESULTS Carbonic anhydrase actiuity: effects of Triton and D T T
The carbonic anhydrase activity of myelin suspended in either water or 0.5% Triton was 4.1 units TABLE1. CONDITIONS FOR
Water 0.5% Triton X-100 Water + DTT 0.5"/, Triton X-100
Extraction of myelin carbonic anhydrase
The extraction of myelin with Triton-DTT is dependent on the concentration of the detergent (Fig. 1). At a concentration of 0.05% Triton, little or no enzyme was solubilized whereas a maximum amount of enzyme is extracted with 1% Triton. Higher concentrations of Triton appear to be inhibitory. The extraction of carbonic anhydrase was also dependent upon the volume of Triton-DTT used per mg of myelin protein. When 3 mg of myelin protein was suspended in l m l 0.5% Triton-DTT, the membrane bound enzyme was solubilized quantitatively by three serial extractions (Fig. 2). Negligible activity remained in the residue. However, if the ratio of protein to volume of Triton-DTT was doubled, more than a 3-fold drop was observed in the efficiency of the extraction. For the purpose of purification of the enzyme, the following solubilization procedure was found to be most efficient: myelin was extracted with 0.05% Triton-DTT followed by a single extraction with 0.5% Triton-DTT. The first extraction removed non-enzymatic protein with no loss of enzyme. The subsequent extraction with 0.5% Triton-DTT solubilized more than 75% of the enzyme and resulted in close to a 50-fold increase in specific activity to a value of 544
ASSAY AND SOLUBILIZATION OF MYELIN CARBONIC: ANHYDRASE
Total units*
Assay condition a. b. c. d.
per mg protein (Table 1). When myelin was suspended in DTT prior to assay, the activity was increased 3-fold regardless of the presence or absence of Triton. Almost half of the enzyme activity was solubilized by a single extraction with Triton-DTT and advantage was taken of this observation for the purification of the myelin carbonic anhydrase (described below). O n the other hand, only a small portion of the enzyme was solubilized by Triton alone. If the Tritonsoluble fraction is incubated in DTT prior to assay, a 3-fold increase in activity is again observed. The activity of the Triton-soluble fraction, even when assayed in the presence of DTT, is significantly less than the activity of the Triton-DTT soluble fraction. These observations suggest that DTT facilitates the dissociation of the enzyme from the- membrane but that there is an additional effect of DTT on the activity which is independent of its effect on solubilization.
+ DTT
* From 14.6 mg myelin protein. t Myelin was extracted once at a
Units/mg protein
60
4.1
60 182 182
4.1 12.5 12.5
Total units solubilizedt by Triton Triton + DTT
16 50
85
protein concentration of 3.3 mg/ml with either Triton or Triton f DTT. The material solubilized with Triton alone was assayed either without (b) or with (d) added DTT
Myelin carbonic anhydrase
70 60
507
-
25
FIG.2. Serial extraction of myelin with 0.50,; Triton X-100 + 0.1% DTT. Myelin was extracted as described in Fig. 1 and in Methods. The myelin residuc was resuspended in Triton X-100 + DTT prior to assay.
TRITON CONCENTRATION (vol. %)
Frc. 1. Extraction of myelin carbonic anhydrase with increasing amounts of Triton X-100. Myelin (3.3mg protein/ml) was extracted at room temperature for 20 min by periodic homogenization in a Dounce homogenizer in the presence of 0.1% dithiothreitol. Solubilization was assessed by assay of the supernatant after centrifugation at 30,000 g for 15 min.
washing step removes non-specifically bound protein and maintains the column at pH 8.0 which is required for maximum binding of the eluting inhibitor in the following step. Subsequent elution of the column with M-acetazolamide reproducibly results in an enzyme preparation with a specific activity between 900 and 2000 units/mg protein corresponding to over a 100-fold purification. The homogeneity of the myelin carbonic anhydrase preparation is suggested by the single band observed on SDS gels when relatively large amounts of protein are used (Fig. 4). The molecular weight of the purified myelin carbonic anhydrase corresponds to 30,000 daltons since it migrates in SDS gels to the same position as the soluble red blood cell enzyme. Upon lyophilization, the purified enzyme loses both activity and solubility. As a consequence, electrophoresis in non SDS gels has not been possible. This is in contrast to myelin which can be lyophilized without loss of activity (CAMMER et al., 1976; Sapirstein & Lees, unpublished). The amino acid composition is characterized by relatively large amounts of aspartic acid, glutamic acid, serine, alanine and glycine (Table 3). Dicarboxylic acids account for 19.6% of the total amino acids recovered whereas basic amino acids account for only 12.9%. The cysteine content, based on recovery of cysteic acid is between one and two residues per mol. The protein is particularly low in S-containing and in aromatic amino acids.
units per mg protein (Table 2). Carbonic anhydrase has been enriched in this fraction to such an extent that a band corresponding to soluble carbonic anhydrase is clearly visible on gels of the 0.5% Triton-DTT extract whereas it is not detected on gels of the original myelin (Fig. 3). AfJinity chromatography
Preliminary studies showed that red blood cell carbonic anhydrase can be quantitatively adsorbed to PAMBS-Sepharose CL-4B in the presence of Triton and can be dissociated from the matrix with high concentrations of a soluble inhibitor. Advantage was taken of this observation to purify the solubilized carbonic anhydrase. The Triton-DTT solubilized fraction of the myelin is chromatographed on a PAMBSSepharose column pre-equilibrated with Triton-DTT. Carbonic anhydrase activity is retained on the column and is not eluted with either Triton-DTT or Triton-DTT diluted 1: 10 and containing 50 mu-Tris sulfate pH 8.0, (Table 2). The gel pattern of the column eluate reflects the selective deletion of the carbonic anhydrase band (Fig. 3-gel 3). The Tris sulfate TABLE2. PURlFlCATloN OF
MYELIN CARBONIC ANHYDRASE
Total units
Myelin 0.05% Triton-DTT extract 0.5% Triton-DTT extract Sulfonamide affinity Column* (1) Triton-DTT eluate (2) Triton-DTT + acetazolamide eluate
Protein (mg)
Specific activity
1050
98 1.90 1.93
13.7 44 544
39 500
1.80 0.24
21.7 2083
1340 84
Yield
% -
6.3 78
2.9 31.3
* PAMBS-Sepharosecolumn eluted with (1) 0.5% Triton-0.17; DTT and then washed with NaCl-Tris sulfate buffer in Triton-DTT as described under M-acetazolamide. Methods and (2) same but containing
VICTORS. SAPIRSTEIN and MARJORIE B. LEES
508 TABLE3. AMINOA C I D
COMPOSITION OF MYELIN CARBONIC ANHYDRASE
mol percent I 2
Amino acid Aspartic Threonine Serine Glutamic Proline Glycine
9.5 5.6 12.1 10.1 4.6 12.6 8.8 0.8 5.4
Alanine
112 Cystine* Valine
.o
Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
* Determined
1
4.0 7.3 2.0 3.7 3.4 6.3 3.2
9.3 4.6 11.6 10.8 4.9 13.1 8.5 ~
5.5 1.2 3.9 7.2 2.6 3.4 3.4 6.4 3.3
as cysteic acid.
DISCUSSION
This paper describes the first purification of a membrane bound carbonic anhydrase not only from brain but from any tissue. The procedure is simple and rapid and can be carried out on myelin obtained from a single rat brain. It should therefore be useful for metabolic studies on the regulation of myelin carbonic anhydrase. The hundred-fold increase in specific activity shown in Table 2 may, in fact, be an underestimation of the extent of purification. After affinity chromatography the specific activity increases only 4-fold over that of the solubilized preparation. However, the gel electrophoretic patterns of these fractions suggest greater purification. The specific activity of the purified enzyme may be low either because of incomplete removal of the inhibitor used to elute the columns or because of a requirement of the enzyme for lipid. At present there is insufficient data to distinguish between these possibilities. An important determinant of enzyme activity appears to be the maintenance of cysteine in the reduced form. Dithiothreitol has a significant effect on myelin carbonic anhydrase activity independent of whether the enzyme is membrane bound or solubilized. Therefore DTT seems to have a direct effect on the enzyme which is not mediated via effects on the intact membrane structure. Carbonic anhydrase from other tissues contain a single cysteine and the myelin enzyme is similar with a cysteine content of one to two residues (1.5 residues based on a molecular weight of 30,000). The marked increase in activity expressed after reduction with DTT suggests that the oxidation-reduction of cysteine could provide a physiologic control mechanism. The amino acid composition is characterized by relatively large amounts of dicarboxylic amino acids, glycine. alanine and serinc. To the best of our knowl-
edge the composition of rat brain soluble carbonic anhydrase has not been reported but the values we obtained are similar to those found in soluble carbonic anhydrases from other species (CARTER1972). Carbonic anhydrase can exist as at least two different isozymes and the high serine content is typical of the B isozyme. However, the myelin enzyme is unaffected by imidazole (SAPIRSTEIN& FLYNN,in preparation), a specific inhibitor of the B isozyme (KANNANet al., 1977). On the other hand, the solubilized myelin enzyme is refractory to chloride (SAPIRSTEINet al., 1977), a property which is typical of the C isozyme. Thus, the myelin enzyme appears to correspond to a C type isozyme. It is therefore similar to the soluble brain carbonic anhydrase which has been characterized in humans on the basis of its immunologic cross reactivity with the C isozyme from erythrocytes (FUNAKOSHI & DELJTSCH 1971). The high serine content observed in our preparations may therefore be a consequence of the presence of lipid serine. MCKINLEY & WHITNEY(1976) have suggested that the membrane bound carbonic anhydrase from kidney may be a distinct isozyme. A comparison of the soluble and myelin forms of the enzyme from brain requires purification of the respective proteins and we are currently purifying the soluble enzyme from brain. Ackrzowledge,nent--The study was supported in part by U.S. Public Health Service Grant NS 13649 and HD 05 155.
REFERENCES BERCMEYER H. U., BERNTE. & NESS B. (1965) in Methods of Enzymatic Analysis (BERGMEYER H. U., ed.). pp. 736-741. Academic Press, New York. BOURKER. S., KIMELBERG H. K., WESTC. R. & BREMMER A. M. (1975) J. Neurochem. 25, 323-328. CAMMER W., FREDMAN T., ROSE A. L. & NORTONW. T. (1976) J. Neurochem. 27, 165-171. CARTERM. J. (1972) Biol. Rev. 47, 465-513. DAVSON H. & SEGALM. B. (1970) J . Physiol. 209, 131-153. FUNAKOSHI S. & DEUTSCHH. F. (1971) J . biol. Chem. 246, 1088-1092. KANNANK. K., PETEFM., FRIDBERG K., CID-DRESDNER H . & HOVGREN S. (1977) FEBS Lelr. 73, 115-126. KARLER S. & WOODBURYD. M. (1960) Biochem. J . 75, 538-543. LEESM. B. & PAXWAN S. A. (1972) Anirlyt. Eiochem. 47, 184-1 92. LOWRY0. H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) J. biol. Chem. 193, 265-275. MARENT. H. (1960) J. Pharmac. exp. Ther. 130, 26-29. MARENT. H. (1967) Physiol. Rel;. 47, 595-781. MCKIVLEYD. N. & WHITNEYP. L. (1976) Biochim. biophy.7. Acra 445, 780-790. NORTONW. T. & PODUSLOS. (1973) J. Neurochem. 21, 749- 7 5 7. SAPIRSTEIN V. S. & LEES M. B. (1977) Trans. Am. Soc. Ncurncbcm. 8, 57 I . SAPIRSTFIN V. S.. TRACHTENHERG M. C., LEES M. B. & KOUL 0. in M \dinarion and Drn?yelinarion: Rccenr
509
FIG. 3. Purification of myelin carbonic anhydrase from one rat brain monitored by polyacrylamide gel electrophoresis. Gel 1, whole rat brain myelin, lOOpg protein; gel 2, material solubilized by Triton, 40pg protein; gel 3, material freely eluted from PAMBS Sepharose, S O p g protein; gel 4, material obtained after elution with acetazolamide: and gel 5, purified carbonic anhydrase standard, 25 pg protein. Electrophoresis was performed as described in Methods. All samples were added in a SO pI volume of 1%. SDS-O.I% DTT-4”/, sucrose. The anode is at the bottom of the figure. FIG.4. Comparison of polyacrylamide gels of purified myelin carbonic anhydrase and soluble carbonic anhydrase C. Gel 1, myelin carbonic anhydrase, 5 O p g protein and gel 2, soluble carbonic anhydrase C. 5 0 p g protein. Conditions as in Fig. 3.
Myelin carbonic anhydrase Chemictrl Adrarices (PALO J . & RIEKLNNEN P., eds.). Plenum Press. New York. In press. WEBER K. & OSBORNM. (1969) J. biol. Chem. 244, 4406-44 12. WHITNEY P. L. (1974) Ano/yr. Biorhem. 57, 467-476.
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YANURASITZ J . R.. ERNSTS. A. & SALGANICOFF L. (1976) J. Neurochem. 27. 707-71 5. ZAiiLm W. L. (1971) In Merhods in Enzymoloqy Vol. XXXII. (FLEISCHER s. & PACKER L.,eds.) pp. 70-81. Academic Press. New York.
