ANALYTICAL
BIOCHEMISTRY
Purification
95, 2-7 (1979)
of Phosphotransacetylase
by Affinity
LINDATOMBRASSMMITH~ANDNATHAN University of California -San
Chromatography1
O.KAPLAN
Diego, Department of Chemistry, Q-058, La Jolla, California 92093 Received October 1978
Phosphotransacetylase from Clostridium kluyveri was purified using a Cs-(6-aminohexyl)amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-foId and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5’-dithiobis(2nitrobenzoic acid) is described.
Phosphotransacetylase (EC 2.3.1.8, acetyl CoA:orthophosphate acetyltransferase) from Clostridium kluyveri was first studied by Stadtman (l-3), who was able to obtain the enzyme in partially pure form. More recently, the transacetylase was purified to apparent homogeneity by conventional methods (4). However, the lengthy procedure resulted in a low yield of enzyme. In view of the remarkable success of affinity chromatography in the purification of enzymes, attempts were carried out to purify PTA3 by this method. It has been established that desulfo-coenzyme A (coenzyme A in which the terminal sulfhydryl group is replaced by a hydrogen atom) is a potent inhibitor of PTA, KI = 4.0 x IO+ M (5). Therefore, the CoA analog was considered to be a potentially suitable ligand for affinity chromatography. In this report the synthesis and use of Sepharose-bound desulfo-CoA in the purification of phosphotransacetylase are described. In addition, a method for the
determination of the purity of PTA by activity staining of polyacrylamide gels is introduced. Preliminary data from this work have been published elsewhere (6). MATERIALS
Materials. Sepharose 4B was purchased from Pharmacia. Diethylaminoethyl-cellulose (DE 11) was purchased from Whatman. Raney nickel was obtained from ICN-K and K Laboratories, Inc. 5,5’-Dithiobis(2nitrobenzoic acid) was obtained from Aldrich Chemical Company. Acetyl phosphate was purchased from Sigma. Commercially purified phosphotransacetylase was obtained from Boehringer Mannheim. A frozen cell paste of Clostridium kluyveri was kindly provided by G. D. Novelli. Reduced coenzyme A was a gift of P-L Biochemicals, Inc. Acetyl coenzyme A was synthesized by the method of Simon and Shemin as described by Stadtman (7), in which CoA is reacted with cold aqueous acetic anhydride. There were two modifications of this method: (i) the reaction was carried out in ammonium bicarbonate buffer at pH 8; and (ii) the extent of reaction was monitored by Ellman’s method for the determination of thiols (8). Preparation of Sepharose-bound desulfo-
1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 Present address: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pa. 19104. 3 Abbreviations used: PTA, phosphotransacetylase; CoA, coenzyme A; DEAE, diethylaminoethyl; DTNB, 5’,5’-dithiobis(2-nitrobenzoic acid). 0003~2697/79/070002-06$02.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
2
PURIFICATION
coenzyme
OF PHOSPHOTRANSACETYLASE
A. The sulfur atom of coenzyme
A was removed with Raney nickel according to the method of Chase et al. (9). A 40-ml slurry of the catalyst was used per gram of CoA. The course of the reaction was monitored by the use of the DTNB (8). When the reaction was completed, the nickel was removed by centrifugation and the solution was applied to a DEAE-cellulose column (3 x 25 cm) that had been equilibrated to pH 8 with ammonium bicarbonate and washed with water. Desulfo-CoA was eluted from the column with a 2-liter gradient (O0.4 M NH,HC03). Fractions having an absorbance maximum at 257 nm were pooled and lyophilized. The presence of desulfoCoA was established by nuclear magnetic resonance. CR-(6-Aminohexyl)-amino-desulfo-CoA was prepared via bromination by the method of Lee ef al. (10) with slight modification. The reaction mixture was buffered at pH 4.5 and the reaction volume was 5 ml/500 mg of desulfo-CoA. The elution gradient for ion-exchange chromatography was 0 to 0.4 M NH&O,. A 200-ml DEAE-cellulose column was used. The ammonium salt of C8-bromodesulfo-CoA was reacted with 1,6diaminohexane according to the method of Lee et uf. (10); however, the reaction took place overnight at room temperature. The reaction mixture was diluted lo-fold and added to a lOO-ml column of DEAE-cellulose (bicarbonate form, pH 8). The product was eluted with a 0 to 0.2 M NH,CO, gradient. Fractions that exhibited an absorbance maximum at 278 nm were pooled and lyophilized. C8-(6-Aminohexyl)-amino-desulfo-CoA was coupled to Sepharose by the CNBr method of Cuatrecasas (11) using 4 mg of derivatized CoA per milliliter of packed Sepharose. After coupling, the gel was collected by suction filtration and washed. Ultraviolet analysis (278 nm) of the filtrate indicated that the ligand density was approximately 0.5 pmol/ml of Sepharose. Puri$cation procedure for phosphotransacetylase. A frozen cell paste of Ciostrid-
3
was diluted 1:5 (w/v) with 10 mM Tris-HCl buffer, pH 8, containing 3 mM dithioerythritol. The suspended cells were disrupted by sonication in an MSE ultrasonic disintegrator, Instrumentation Associates, at 200 W and 20 kHz. The suspension was sonicated for ten 2-min intervals with intermittent cooling to 6°C. The sonicate was centrifuged at 30,900# for 20 min and the pellet was discarded. Two milliliters of the supernatant solution were brought to 40% saturation with ammonium sulfate by the slow addition, with stirring, of solid ammonium sulfate. The mixture was stirred for 15 min and centrifuged at 27,OOOg for 20 min. The pellet was discarded and the supernatant solution was brought to 60% saturation with ammonium sulfate by the addition of solid ammonium sulfate. The mixture was again stirred and centrifuged and the supernatant solution was discarded. The pellet was brought to 1 ml by the addition of 0.1 M Tris-HCl, pH 8.0. The solution obtained from the ammonium sulfate fractionation was desalted by passage through a Sephadex G-25 column (1.2 x 18 cm) preequilibrated with 10 mM KH2P04, pH 6.5. Fractions containing activity were pooled (approximately 4 ml), diluted to 10 ml with buffer, and added to the desulfo-CoA-Sepharose column (2-ml bed volume) preequilibrated with 10 mM KH2P04, pH 6.5. After the addition of the protein, the column was washed with equilibration buffer until the absorbance of the effluent at 280 nm was less than 0.05 OD units. The activity was eluted with a 40-ml gradient from 0 to 0.5 mM CoA containing 10 mM KH2P04, pH 6.5. The flow rate was 0.33 ml/mm and 2-ml fractions were collected. Phosphotransacetylase activity was determined by a modification of the method of Bergmeyer et ul. (4). The increase in absorption at 233 nm due to the formation of acetyl CoA from acetyl phosphate and CoA was measured. The activity was determined
ium kluyveri
4
SMITH AND KAPLAN
in a total volume of 1 ml containing 90 mM Tris-HCl, pH 8, 10 mM ammonium sulfate, 2 mM acetyl phosphate, and 0.13 mM CoA. One unit of activity is defined as 1 pmol of acetyl CoA formed per minute. Protein concentration was determined by the use of fluorescamine (12). Polyacrylamide gel electrophoresis. Polyacrylamide gels were prepared and electrophoresis was carried out by the method of Gabriel (13) with minor modification. Each gel contained the following: 8% acrylamide; 0.21% N,N’-methylene-bisacrylamide; 0.15% N,N,N’,N’-tetramethylenediamine (v/v); 0.08% ammonium persulfate; and 0.375 M Tris-HCl, pH 8.8. Sample and stacking gels were not used. Gels were run at 1 to 3 mA per gel at room temperature. When electrophoresis was completed, the gel was stained for activity. First, the gel was rinsed two times with water and soaked in 0.1 M KHzPOl, pH 7, for 15 min. Then the buffer was discarded and the gel was incubated with 3.2 ml of substrate solution which contained 100 pmol of ammonium sulfate and 2.3 pmol of acetyl CoA in 94 mM KH2POI, pH 7. After a 5-min incubation in the substrate mixture, 0.050 of a 10 mM solution of DTNB was added. To record the position of the activity band the gel was scanned at 412 nm using a Gilford gel scanner equipped with a Beckman DU light source and monochrometer. Immediately after the gel was scanned, its length was measured to the nearest 0.5 mm, rinsed twice with distilled water, and stained for protein TABLE PURIFICATION
Procedure Crude extract Desalted ammonium sulfate fractionation (40-60%) Affinity chromatography
Volume (ml) 2 10 4
in a solution of 0.2% Coomassie brilliant blue in 50% trichloroacetic acid (w/v). The gel was destained in acetic acid:ethanol: water (75:50:875). After destaining was completed, the length of the gel was adjusted to that recorded after the activity scan by incubating the gel in 12.5% ethanol. At this point, the gel was scanned for protein at 600 nm. The purity of phosphotransacetylase was quantitatively evaluated by integration. RESULTS
The results of the purification procedure are shown in Table 1. The crude extract was first enriched with PTA activity by an ammonium sulfate fractionation. This step generally yielded 85-90% of the original activity. After fractionation, the enzyme was desalted over a Sephadex G-25 column, with virtually total recovery. Between 2500 and 3000 units of desalted enzyme was added to the affinity gel. From 5 to 20% of the activity washed through initially, with less activity binding after several uses of the column. It was apparent that the affinity gel gradually lost its capacity to bind the transacetylase. Perhaps the loss of capacity was caused by the mechanical breakdown of the gel or by catalytic hydrolysis of the ligand by enzymes in the extract or by irreversible binding of proteins to the column material. After the initial wash of activity, low levels of activity continued to leak through under the conditions used to wash nonspecific proteins off the 1
OF PHOSPHOTRANSACETYLASE
Total protein (mid
Total activity (units)
56
2770
17 0.10
2478 714
Specific activity (units/mg) 49.5 146 7140
Yield (%I
Purification (n-fold)
100
-
89.5 25.8
2.9 144
PURIFICATION
OF PHOSPHOTRANSACETYLASE
Fraction
5
Number
FIG. 1. Affinity chromatography of phosphotransacetylase using Sepharose-bound Waminohexyl)-amino-desulfo-CoA. The arrow indicates the start of a 0 to 0.5 mM CoA gradient in 10 mM KH,PO,, pH 6.5. Protein (x) and phosphotransacetylase activity (0) were monitored.
column. This leakage seemed to be independent of the age of the column. The activity was eluted with a CoA gradient. As can be seen in Fig. 1, PTA was eluted in a very sharp peak at the onset of the gradient with an addition of approximately 50 PM CoA. Typically, between 50 and 70% of the initial activity was recovered in the elution peak. However, only the fractions of the highest specific activity were pooled, thus reducing the yield to about 30%. The total activity recovered from the column, including all fractions, was about 80%. The protein was purified about 50-fold by affinity chromatography. The increase in specific activity produced by affinity chromatography was identical whether a crude extract or the ammonium sulfate fractionated enzyme was added to the affinity matrix. Therefore, an enriched extract was used for affinity chromatography to obtain a higher final specific activity. Gel filtration over a Sephadex G-75 column before or after affinity chromatography did not increase the final specific activity over that reported in Table 1. Using this three-step procedure, the final specific activity is over 7000 units/ mg with an overall yield of 25%. Phosphotransacetylase from C. kluyveri
is commercially available as an ammonium sulfate precipitate at a specific activity of 1000 units/mg. An attempt to purify this commercial preparation failed because dialysis or dilution of the enzyme was required in order to prepare it for affinity chromatography. It has been found that these procedures inactivate the enzyme (3). To measure the purity of the PTA preparations polyacrylamide gel electrophoresis was carried out. The transacetylase activity in gels was determined by the measurement of free CoA formed from acetyl CoA and phosphate ion. The production of free CoA in the gel was detected with DTNB as a yellow band. DTNB did not interfere in the activity assay because the presence of acetyl CoA completely protects the enzyme against inactivation by DTNB (14). An activity band appeared only when a gel containing PTA activity was incubated with both substrates in the presence of DTNB. The results of electrophoresis of phosphotransacetylase purified in this study are given in Fig. 2. The protein was found to be about 73% pure. The commercial preparation of PTA was submitted to gel electrophoresis as well (Fig. 3). The activity peak coincides with
6
SMITH
AND KAPLAN
the smallest detectable protein peak. By integration of the protein peaks, this preparation was found to be about 6% pure. Other lots of the commercial preparation were found to be up to 8% pure by this method. Due to the low purity of the commercial preparation (1000 units/mg), reports in the literature on the properties of the enzyme using these preparations should be interpreted with caution (see 14). DlSCUSSlON
It has been shown that the utilization of desulfo-CoA-Sepharose affinity chromatography in conjunction with ammonium sulfate fractionation is highly successful in purifying phosphotransacetylase. Bergmeyer ei al. (4) purified the enzyme 83-fold to a specific activity of 9100 units/mg by conventional methods. The enzyme appeared to be homogeneous. However, this product was obtained only after a IO-step procedure and with an 8% yield. The specific activity obtained by the procedure detailed in this report was about 20% lower than that obtained by Bergmeyer et al.
b
FIG. 2. Polyacrylamide gel electrophoresis of purified phosphotransacetylase. Five micrograms of protein were electrophoresed. The gel was scanned for protein (a) and activity (b).
b 0 15
cE 010 N d 005 :
0
--5Distance
4 (cm)
6
FIG. 3. Polyacrylamide gel electrophoresis of commercial phosphotransacetylase. Fifteen micrograms of protein were submitted to electrophoresis. The gel was scanned for protein (a) and activity (b).
Nevertheless, by using the affinity chromatography procedure the yield was over three times higher, and the method is much less cumbersome than the conventional method. In addition, increasing the volume of the affinity matrix to 20 ml should increase the yield of phosphotransacetylase to the milligram range. A similar methodology may be used to purify other CoA-requiring enzymes. Desulfo-CoA will likely be an excellent inhibitor of other CoA enzymes. Desulfo-CoA is a good analog of CoA to use for immobilization because it lacks the sulfhydryl group which would interfere with the bromination. Moreover, the affinity gel itself may be regenerated after use without the added complication of reducing the sulfhydryl group of CoA. As described above, the purity of PTA
PURIFICATION
OF PHOSPHOTRANSACETYLASE
may be determined by activity staining of polyacrylamide gels with DTNB. DTNL. The 1 -.. pk-ity of other enzymes that catalyze the t deesterification of CoA may be determined determl by a modification of this method of staining. ar ACKNOWLEDGMENTS The authors wish to acknowledge Dr. C. Y. Lee for his help with the bromination of desulfo-CoA. This study was supported by grants from the U. S. Public Health Service (USPHS CA 11683) and the American Cancer Society (BC-60-R).
REFERENCES 1. Stadtman, E. R. (1952) .I. Bid. Chem. 196, 527534. 2. Stadtman, E. R. (1952) .I. Bid. Chem. 196, 535546. 3. Stadtman, E. R. (1955) in Methods in Enzymology (Colowick. S. P., and Kaplan, N. 0.. eds.), Vol. 1, pp. 596-599, Academic Press, New York. 4. Bergmeyer, H. U., Holz, G., Klotzsch, H., and Lang, G. (1963) Biochem. Z. 338, 114-121.
7
5. Shimizu, M., Suzuki, T.. Hosokawa, Y., Nagase. 0.. and Abiko. Y. (1970) Biochim. Bioph~s. Acta 222, 307-319. 6. Lee, C. Y., and Johansson. C. J. (1977) And. Biochrm. 77, 90- 102. 7. Stadtman. E. R. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.). Vol. 3. pp. 931-941, Academic Press. New York. 8. Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70-77. 9. Chase, J. F. A., Middleton, B., and Tubbs. P. K. (1966) Biochem. Biophys. RPS. Cr,mmun. 23, 208-213. 10. Lee, C. Y., Lappi, D.A., Wermuth. B.. Everse, J.. and Kaplan. N. 0. (1974) Arch. Biochrm. Biophys. 163, 561-569. 11. Cuatrecasas, P. ( 1970) J. Bid. Chum. 245. 30593065. 12. Bohlen, P., Stein, S., Dairman, W.. and Udenfriend. S. (1973) Arch. B&hem. Biophys. 155, 213-220. 13. Gabriel. 0. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 565-578. Academic Press, New York. 14. Henkin, J.. and Abeles, R. H. (1976) Bioc,hemistry 15, 3472-3479.
BIOCHEMISTRY
Purification
95, 2-7 (1979)
of Phosphotransacetylase
by Affinity
LINDATOMBRASSMMITH~ANDNATHAN University of California -San
Chromatography1
O.KAPLAN
Diego, Department of Chemistry, Q-058, La Jolla, California 92093 Received October 1978
Phosphotransacetylase from Clostridium kluyveri was purified using a Cs-(6-aminohexyl)amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-foId and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5’-dithiobis(2nitrobenzoic acid) is described.
Phosphotransacetylase (EC 2.3.1.8, acetyl CoA:orthophosphate acetyltransferase) from Clostridium kluyveri was first studied by Stadtman (l-3), who was able to obtain the enzyme in partially pure form. More recently, the transacetylase was purified to apparent homogeneity by conventional methods (4). However, the lengthy procedure resulted in a low yield of enzyme. In view of the remarkable success of affinity chromatography in the purification of enzymes, attempts were carried out to purify PTA3 by this method. It has been established that desulfo-coenzyme A (coenzyme A in which the terminal sulfhydryl group is replaced by a hydrogen atom) is a potent inhibitor of PTA, KI = 4.0 x IO+ M (5). Therefore, the CoA analog was considered to be a potentially suitable ligand for affinity chromatography. In this report the synthesis and use of Sepharose-bound desulfo-CoA in the purification of phosphotransacetylase are described. In addition, a method for the
determination of the purity of PTA by activity staining of polyacrylamide gels is introduced. Preliminary data from this work have been published elsewhere (6). MATERIALS
Materials. Sepharose 4B was purchased from Pharmacia. Diethylaminoethyl-cellulose (DE 11) was purchased from Whatman. Raney nickel was obtained from ICN-K and K Laboratories, Inc. 5,5’-Dithiobis(2nitrobenzoic acid) was obtained from Aldrich Chemical Company. Acetyl phosphate was purchased from Sigma. Commercially purified phosphotransacetylase was obtained from Boehringer Mannheim. A frozen cell paste of Clostridium kluyveri was kindly provided by G. D. Novelli. Reduced coenzyme A was a gift of P-L Biochemicals, Inc. Acetyl coenzyme A was synthesized by the method of Simon and Shemin as described by Stadtman (7), in which CoA is reacted with cold aqueous acetic anhydride. There were two modifications of this method: (i) the reaction was carried out in ammonium bicarbonate buffer at pH 8; and (ii) the extent of reaction was monitored by Ellman’s method for the determination of thiols (8). Preparation of Sepharose-bound desulfo-
1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 Present address: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pa. 19104. 3 Abbreviations used: PTA, phosphotransacetylase; CoA, coenzyme A; DEAE, diethylaminoethyl; DTNB, 5’,5’-dithiobis(2-nitrobenzoic acid). 0003~2697/79/070002-06$02.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
2
PURIFICATION
coenzyme
OF PHOSPHOTRANSACETYLASE
A. The sulfur atom of coenzyme
A was removed with Raney nickel according to the method of Chase et al. (9). A 40-ml slurry of the catalyst was used per gram of CoA. The course of the reaction was monitored by the use of the DTNB (8). When the reaction was completed, the nickel was removed by centrifugation and the solution was applied to a DEAE-cellulose column (3 x 25 cm) that had been equilibrated to pH 8 with ammonium bicarbonate and washed with water. Desulfo-CoA was eluted from the column with a 2-liter gradient (O0.4 M NH,HC03). Fractions having an absorbance maximum at 257 nm were pooled and lyophilized. The presence of desulfoCoA was established by nuclear magnetic resonance. CR-(6-Aminohexyl)-amino-desulfo-CoA was prepared via bromination by the method of Lee ef al. (10) with slight modification. The reaction mixture was buffered at pH 4.5 and the reaction volume was 5 ml/500 mg of desulfo-CoA. The elution gradient for ion-exchange chromatography was 0 to 0.4 M NH&O,. A 200-ml DEAE-cellulose column was used. The ammonium salt of C8-bromodesulfo-CoA was reacted with 1,6diaminohexane according to the method of Lee et uf. (10); however, the reaction took place overnight at room temperature. The reaction mixture was diluted lo-fold and added to a lOO-ml column of DEAE-cellulose (bicarbonate form, pH 8). The product was eluted with a 0 to 0.2 M NH,CO, gradient. Fractions that exhibited an absorbance maximum at 278 nm were pooled and lyophilized. C8-(6-Aminohexyl)-amino-desulfo-CoA was coupled to Sepharose by the CNBr method of Cuatrecasas (11) using 4 mg of derivatized CoA per milliliter of packed Sepharose. After coupling, the gel was collected by suction filtration and washed. Ultraviolet analysis (278 nm) of the filtrate indicated that the ligand density was approximately 0.5 pmol/ml of Sepharose. Puri$cation procedure for phosphotransacetylase. A frozen cell paste of Ciostrid-
3
was diluted 1:5 (w/v) with 10 mM Tris-HCl buffer, pH 8, containing 3 mM dithioerythritol. The suspended cells were disrupted by sonication in an MSE ultrasonic disintegrator, Instrumentation Associates, at 200 W and 20 kHz. The suspension was sonicated for ten 2-min intervals with intermittent cooling to 6°C. The sonicate was centrifuged at 30,900# for 20 min and the pellet was discarded. Two milliliters of the supernatant solution were brought to 40% saturation with ammonium sulfate by the slow addition, with stirring, of solid ammonium sulfate. The mixture was stirred for 15 min and centrifuged at 27,OOOg for 20 min. The pellet was discarded and the supernatant solution was brought to 60% saturation with ammonium sulfate by the addition of solid ammonium sulfate. The mixture was again stirred and centrifuged and the supernatant solution was discarded. The pellet was brought to 1 ml by the addition of 0.1 M Tris-HCl, pH 8.0. The solution obtained from the ammonium sulfate fractionation was desalted by passage through a Sephadex G-25 column (1.2 x 18 cm) preequilibrated with 10 mM KH2P04, pH 6.5. Fractions containing activity were pooled (approximately 4 ml), diluted to 10 ml with buffer, and added to the desulfo-CoA-Sepharose column (2-ml bed volume) preequilibrated with 10 mM KH2P04, pH 6.5. After the addition of the protein, the column was washed with equilibration buffer until the absorbance of the effluent at 280 nm was less than 0.05 OD units. The activity was eluted with a 40-ml gradient from 0 to 0.5 mM CoA containing 10 mM KH2P04, pH 6.5. The flow rate was 0.33 ml/mm and 2-ml fractions were collected. Phosphotransacetylase activity was determined by a modification of the method of Bergmeyer et ul. (4). The increase in absorption at 233 nm due to the formation of acetyl CoA from acetyl phosphate and CoA was measured. The activity was determined
ium kluyveri
4
SMITH AND KAPLAN
in a total volume of 1 ml containing 90 mM Tris-HCl, pH 8, 10 mM ammonium sulfate, 2 mM acetyl phosphate, and 0.13 mM CoA. One unit of activity is defined as 1 pmol of acetyl CoA formed per minute. Protein concentration was determined by the use of fluorescamine (12). Polyacrylamide gel electrophoresis. Polyacrylamide gels were prepared and electrophoresis was carried out by the method of Gabriel (13) with minor modification. Each gel contained the following: 8% acrylamide; 0.21% N,N’-methylene-bisacrylamide; 0.15% N,N,N’,N’-tetramethylenediamine (v/v); 0.08% ammonium persulfate; and 0.375 M Tris-HCl, pH 8.8. Sample and stacking gels were not used. Gels were run at 1 to 3 mA per gel at room temperature. When electrophoresis was completed, the gel was stained for activity. First, the gel was rinsed two times with water and soaked in 0.1 M KHzPOl, pH 7, for 15 min. Then the buffer was discarded and the gel was incubated with 3.2 ml of substrate solution which contained 100 pmol of ammonium sulfate and 2.3 pmol of acetyl CoA in 94 mM KH2POI, pH 7. After a 5-min incubation in the substrate mixture, 0.050 of a 10 mM solution of DTNB was added. To record the position of the activity band the gel was scanned at 412 nm using a Gilford gel scanner equipped with a Beckman DU light source and monochrometer. Immediately after the gel was scanned, its length was measured to the nearest 0.5 mm, rinsed twice with distilled water, and stained for protein TABLE PURIFICATION
Procedure Crude extract Desalted ammonium sulfate fractionation (40-60%) Affinity chromatography
Volume (ml) 2 10 4
in a solution of 0.2% Coomassie brilliant blue in 50% trichloroacetic acid (w/v). The gel was destained in acetic acid:ethanol: water (75:50:875). After destaining was completed, the length of the gel was adjusted to that recorded after the activity scan by incubating the gel in 12.5% ethanol. At this point, the gel was scanned for protein at 600 nm. The purity of phosphotransacetylase was quantitatively evaluated by integration. RESULTS
The results of the purification procedure are shown in Table 1. The crude extract was first enriched with PTA activity by an ammonium sulfate fractionation. This step generally yielded 85-90% of the original activity. After fractionation, the enzyme was desalted over a Sephadex G-25 column, with virtually total recovery. Between 2500 and 3000 units of desalted enzyme was added to the affinity gel. From 5 to 20% of the activity washed through initially, with less activity binding after several uses of the column. It was apparent that the affinity gel gradually lost its capacity to bind the transacetylase. Perhaps the loss of capacity was caused by the mechanical breakdown of the gel or by catalytic hydrolysis of the ligand by enzymes in the extract or by irreversible binding of proteins to the column material. After the initial wash of activity, low levels of activity continued to leak through under the conditions used to wash nonspecific proteins off the 1
OF PHOSPHOTRANSACETYLASE
Total protein (mid
Total activity (units)
56
2770
17 0.10
2478 714
Specific activity (units/mg) 49.5 146 7140
Yield (%I
Purification (n-fold)
100
-
89.5 25.8
2.9 144
PURIFICATION
OF PHOSPHOTRANSACETYLASE
Fraction
5
Number
FIG. 1. Affinity chromatography of phosphotransacetylase using Sepharose-bound Waminohexyl)-amino-desulfo-CoA. The arrow indicates the start of a 0 to 0.5 mM CoA gradient in 10 mM KH,PO,, pH 6.5. Protein (x) and phosphotransacetylase activity (0) were monitored.
column. This leakage seemed to be independent of the age of the column. The activity was eluted with a CoA gradient. As can be seen in Fig. 1, PTA was eluted in a very sharp peak at the onset of the gradient with an addition of approximately 50 PM CoA. Typically, between 50 and 70% of the initial activity was recovered in the elution peak. However, only the fractions of the highest specific activity were pooled, thus reducing the yield to about 30%. The total activity recovered from the column, including all fractions, was about 80%. The protein was purified about 50-fold by affinity chromatography. The increase in specific activity produced by affinity chromatography was identical whether a crude extract or the ammonium sulfate fractionated enzyme was added to the affinity matrix. Therefore, an enriched extract was used for affinity chromatography to obtain a higher final specific activity. Gel filtration over a Sephadex G-75 column before or after affinity chromatography did not increase the final specific activity over that reported in Table 1. Using this three-step procedure, the final specific activity is over 7000 units/ mg with an overall yield of 25%. Phosphotransacetylase from C. kluyveri
is commercially available as an ammonium sulfate precipitate at a specific activity of 1000 units/mg. An attempt to purify this commercial preparation failed because dialysis or dilution of the enzyme was required in order to prepare it for affinity chromatography. It has been found that these procedures inactivate the enzyme (3). To measure the purity of the PTA preparations polyacrylamide gel electrophoresis was carried out. The transacetylase activity in gels was determined by the measurement of free CoA formed from acetyl CoA and phosphate ion. The production of free CoA in the gel was detected with DTNB as a yellow band. DTNB did not interfere in the activity assay because the presence of acetyl CoA completely protects the enzyme against inactivation by DTNB (14). An activity band appeared only when a gel containing PTA activity was incubated with both substrates in the presence of DTNB. The results of electrophoresis of phosphotransacetylase purified in this study are given in Fig. 2. The protein was found to be about 73% pure. The commercial preparation of PTA was submitted to gel electrophoresis as well (Fig. 3). The activity peak coincides with
6
SMITH
AND KAPLAN
the smallest detectable protein peak. By integration of the protein peaks, this preparation was found to be about 6% pure. Other lots of the commercial preparation were found to be up to 8% pure by this method. Due to the low purity of the commercial preparation (1000 units/mg), reports in the literature on the properties of the enzyme using these preparations should be interpreted with caution (see 14). DlSCUSSlON
It has been shown that the utilization of desulfo-CoA-Sepharose affinity chromatography in conjunction with ammonium sulfate fractionation is highly successful in purifying phosphotransacetylase. Bergmeyer ei al. (4) purified the enzyme 83-fold to a specific activity of 9100 units/mg by conventional methods. The enzyme appeared to be homogeneous. However, this product was obtained only after a IO-step procedure and with an 8% yield. The specific activity obtained by the procedure detailed in this report was about 20% lower than that obtained by Bergmeyer et al.
b
FIG. 2. Polyacrylamide gel electrophoresis of purified phosphotransacetylase. Five micrograms of protein were electrophoresed. The gel was scanned for protein (a) and activity (b).
b 0 15
cE 010 N d 005 :
0
--5Distance
4 (cm)
6
FIG. 3. Polyacrylamide gel electrophoresis of commercial phosphotransacetylase. Fifteen micrograms of protein were submitted to electrophoresis. The gel was scanned for protein (a) and activity (b).
Nevertheless, by using the affinity chromatography procedure the yield was over three times higher, and the method is much less cumbersome than the conventional method. In addition, increasing the volume of the affinity matrix to 20 ml should increase the yield of phosphotransacetylase to the milligram range. A similar methodology may be used to purify other CoA-requiring enzymes. Desulfo-CoA will likely be an excellent inhibitor of other CoA enzymes. Desulfo-CoA is a good analog of CoA to use for immobilization because it lacks the sulfhydryl group which would interfere with the bromination. Moreover, the affinity gel itself may be regenerated after use without the added complication of reducing the sulfhydryl group of CoA. As described above, the purity of PTA
PURIFICATION
OF PHOSPHOTRANSACETYLASE
may be determined by activity staining of polyacrylamide gels with DTNB. DTNL. The 1 -.. pk-ity of other enzymes that catalyze the t deesterification of CoA may be determined determl by a modification of this method of staining. ar ACKNOWLEDGMENTS The authors wish to acknowledge Dr. C. Y. Lee for his help with the bromination of desulfo-CoA. This study was supported by grants from the U. S. Public Health Service (USPHS CA 11683) and the American Cancer Society (BC-60-R).
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5. Shimizu, M., Suzuki, T.. Hosokawa, Y., Nagase. 0.. and Abiko. Y. (1970) Biochim. Bioph~s. Acta 222, 307-319. 6. Lee, C. Y., and Johansson. C. J. (1977) And. Biochrm. 77, 90- 102. 7. Stadtman. E. R. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.). Vol. 3. pp. 931-941, Academic Press. New York. 8. Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70-77. 9. Chase, J. F. A., Middleton, B., and Tubbs. P. K. (1966) Biochem. Biophys. RPS. Cr,mmun. 23, 208-213. 10. Lee, C. Y., Lappi, D.A., Wermuth. B.. Everse, J.. and Kaplan. N. 0. (1974) Arch. Biochrm. Biophys. 163, 561-569. 11. Cuatrecasas, P. ( 1970) J. Bid. Chum. 245. 30593065. 12. Bohlen, P., Stein, S., Dairman, W.. and Udenfriend. S. (1973) Arch. B&hem. Biophys. 155, 213-220. 13. Gabriel. 0. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 565-578. Academic Press, New York. 14. Henkin, J.. and Abeles, R. H. (1976) Bioc,hemistry 15, 3472-3479.
