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Purification of malic enzyme by affinity chromatography on immobilized (N6-(6-aminohexyl)-adenosine 2′,5′-bisphosphate
Purification of Malic Enzyme by Affinity Chromatography on immobilized W-(6-Aminohexyl)-Adenosine 2’,5’-Bisphosphate KWOKKAM Ames Research Laboratory,
YEUNGANDROBERT
J. CARRICO
Ames Company, Division of Miles Laboratories, Elkhart, Indiana 46514
Inc.,
Received November 11, 1975; accepted April 20, 1976 A simple and rapid method for the purification of NADP-dependent malic enzyme from chicken liver is described. A crude tissue extract was chromatographed on an affinity column containing immobilized W-(6arninohexyl)adenosine 2’,5’-bisphosphate. Malic enzyme bound to the ligand and was eluted specifically by a gradient of NADP. Then the enzyme was purified further by gel filtration. The entire procedure requires 50 hr and provides the enzyme in about 40% yield. The purified enzyme migrated as one band during disc electrophoresis in polyacrylamide gel.
NADP-dependent malic enzymes have been isolated from several sources by methods which employ ethanol and ammonium sulfate fractionations, ion-exchange chromatography, and gel filtration (l-3). Although the crystalline enzyme has been prepared (l), the purification procedures are rather time consuming. Brodelius et al. (4) have shown that N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate inhibits several NADP-dependent enzymes, and these were purified by affinity chromatography on an immobilized form of the inhibitor. We have found that the nucleotide derivative also inhibits malic enzyme from chicken liver, and this enzyme can be purified rapidly by affinity chromatography. The purification procedure is of special interest because malic enzyme is used in recently described enzymic-cycling assay for NAD (5). MATERIALS
AND METHODS
The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO.): NAD, NADP, 6-chloropurine riboside, malic acid, glutathione (oxidized), dithiothreitol, phenazine methosulfate, nitroblue tetrazolium, 6-phosphogluconic acid. Lithium lactate, glucose 6-phosphate, NADPH, and a-ketoglutaric acid were purchased from Calbiochem (San Diego, Calif.). Sepharose4B and Sephadex G-200 were from Pharmacia Fine Chemicals (Piscataway, NJ). Glutamic acid and cyanogen bromide were products of Eastman Chemicals (Rochester, N.Y.). 369 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
370
YEUNG
AND
CARRICO
Fresh chicken liver was obtained from a local slaughter house and stored at -20°C until needed. Synthesis of N6-(6-aminohexyl)-adenosine 2’S’-bisphosphate. This synthesis was carried out according to the method of Brodelius et al. (4). The extinction coefficient reported by these authors was used to determine concentrations of the purified nucleotide. Binding of N6-(6-aminohexyl)-adenosine 2 ‘,5 ‘-bisphosphate to Sepharose-4B. One hundred milliliters of Sepharose-4B was activated with cyanogen bromide by the method of March et al. (6). The activated Sepharose was reacted with 455 pmol of the ligand in 100 ml of 0.2 M NaHCO,, pH 9.5, at 4°C for 18 hr. Then the liquid phase was separated by filtration on a sintered-glass funnel, and the optical absorbance at 265 nm was measured. The results indicate that 3.3 pmol of the ligand was bound per milliliter of Sepharose. Enzyme assays. Assays for enzyme activity were conducted at 25°C in 30 mM Tris-HCl buffer, pH 7.4, containing 1 mM dithiothreitol unless other conditions are indicated. Changes in absorbance at 340 nm due to reduction or oxidation of NAD(H) or NADP(H) were measured spectrophotometrically. The substrate and cofactor concentrations employed for the assays of glutamic dehydrogenase, glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase were the same as those used by Brodelius et al. (4). Assays for lactic dehydrogenase were conducted with 0.1 M lithium lactate and 10 mM NAD. Malic enzyme was assayed according to Wise and Ball (7) with some modifications. The reaction mixture contained 66 mM Tris-HCl, pH 7.4, 0.3 mM dithiothreitol, 4 mM MgC&, 0.2 mM NADP, and 0.5 mM malate. A unit of activity reduces 1 pmol of NADP/min. Studies of the inhibition of malic enzyme activity by N6-(6aminohexyl)adenosine 2’,5’-bisphosphate were conducted by measurement of the increase in fluorescence intensity due to reduction of NADP by 1 mM malate. The levels of NADPH and inhibitor were varied and the reaction was initiated by the addition of 0.2 unit of malic enzyme/ml. NADPH oxidase activity in malic enzyme preparations was measured fluorometrically. The reaction solution contained 30 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, and 3 PM NADPH. Protein assay. Measurements of protein were conducted according to the method of Lowry et al. (8), employing bovine serum albumin as the standard. Purification of NADP-dependent malic enzyme. Fifty grams of chicken liver and 150 ml of 30 mM Tris-HCl, pH 7.7, containing 0.25 M sucrose, 1 mM dithiothreitol, and 0.1 mM EDTA were homogenized in a Sorvall Omnimixer for 2 min at 0°C. Particulate material was separated by centrifugation at 30,OOOg for 30 min and then solid (NH&SO, was added
PURIFICATION
OF MALIC
ENZYME
371
to the supernatant to give 20% saturation. The suspension was centrifuged as above and the supernatant was brought to 70% saturation by slow addition of (NH&SO, over a period of 1 hr. The pellet obtained by centrifugation was dissolved in 150 ml of 30 mM Tris-HCI buffer, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. About 70 units of malic enzyme was applied to the affinity column. A 2.5 x 32-cm column of immobilized N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate was equilibrated at 5°C with 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. The crude malic enzyme was passed through the column at 2 ml/min, and then the column was washed with 200 ml of the Tris buffer. Next, the chromatogram was developed with 400 ml of the buffer containing 0.2 M KC1 followed by 300 ml of buffer without KCl. At this point, the absorbance of the effluent at 280 nm was less than 0.03 and a linear gradient of NADP, 0 to 0.5 mM (500 ml total), in 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA was applied at a flow rate of 2 mUmin. The effluent was collected in 6-ml fractions, and these were assayed for enzyme activity. Fractions containing malic enzyme were pooled as described below and concentrated to 4 to 6 ml by pressure dialysis. The concentrated enzyme preparation was applied to a 2.5 x 90-cm column of Sephadex G-200 which was previously equilibrated with 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. The chromatogram was developed with the same buffer and malic enzyme eluted between 30 and 36% of the bed volume. The fractions with more than 0.1 unit of enzyme activity/ml were pooled and concentrated by pressure dialysis. To regenerate the affinity chromatography medium, it was stirred gently in 500 ml of 6 M urea containing 2 M KC1 and filtered on a sintered-glass funnel. Then it was washed with 350 ml of 30 mM Tris-HCl buffer, pH 7.7, containing 1 mM dithiothreitol and 0.1 mrvr EDTA and repacked into a column. The immobilized ligand was used for 10 preparations of malic enzyme without notable change in binding capacity. Disc efectrophoresis. Electrophoresis in 5% polyacrylamide gels was conducted according to the method of Davis (9). Gels were stained for protein with amido black and for malic enzyme activity by the method of Henderson (10). Electrophoresis in the presence of sodium dodecyl sulfate was carried out in 5% polyacrylamide gels by the method of Weber and Osborn (11). RESULTS Purification
of Malic Enzyme
In preliminary studies we subjected a crude chicken liver extract to affinity chromatography after centrifugation at 78,SOOg for 1 hr. The
372
YEUNG AND CARRICO TABLE SPECIFIC
ACITIVITY
AND RECOVERY
1
OF MALIC
ENZYME
DURING
PURIFICATION
Preparation assayed
Enzyme activity (units)
Specific activity (units/mg of protein
Recovery (%I
Liver extract Ammonium sulfate Affinity chromatography Gel filtration
79 70 41 33
0.017 0.019 4.1 7.0
100 88 51 41
chromatography was completed successfully; however, lipid-like material accumulated at the top of the column and reduced the flow rate. The interfering material was separated from the malic enzyme by ammonium sulfate fractionation. Over 80% of the enzyme activity was recovered in the precipitate formed between 20 and 70% saturation with ammonium sulfate (Table 1). A typical elution profile obtained by chromatography of the ammonium sulfate fraction on the affinity column is shown in Fig. 1. Most proteins
l.zj
g 0.6 %04 P 0.2
i 0.6 L0 200 EFFLUENT
VOLUME (ml)
1. Elution profile obtained by chromatography of a crude chicken liver extract on immobilized NB-(6-aminohexyl)-adenosine 2’,5’-bisphosphate. A chicken liver extract was fractionated with ammonium sulfate as described in Materials and Methods. The material precipitated by 70% ammonium sulfate was dissolved in 150 ml of 30 mM Tris-HCI, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA and passed at a flow rate of 2 mUmin into a 2.5 x 32-cm column of Np-(6aminohexyl)-adenosine 2’,5’-bisphosphate bound to Sepharose. At (A) the column was washed with the Tris-HCl buffer, containing 1 mrvt dithiothreitol and 0.1 mM EDTA, and at (B) 0.2 M KC1 was included in the buffer. Then the KC1 was washed from the column with the buffer (C), and finally a linear gradient of NADP was applied at (D). Six-milliliter fractions were collected, and the absorbance at 280 nm (0) was measured in fractions preceding the NADP gradient. The elution profiles for lactic dehydrogenase (0) and malic enzyme (A) are shown. The broken line indicates that NADP concentration. The portion of the chromatogram pooled for the purified malic enzyme preparation is indicated by the horizontal line. FIG.
PURIFICATION
OF MALIC
ENZYME
373
FIG. 2. Disc electrophoresis of purified malic enzyme. Purified malic enzyme was examined by electrophoresis in polyacrylamide gels as described in Materials and Methods. About 20 pg of protein was applied to each gel. Gel (A) was stained for protein with amido black, and gel (B) was stained for malic enzyme activity. Electrophoresis for gel (C) was conducted in the presence of sodium dodecyl sulfate, and the gel was stained with Coomassie blue.
passed through the column unretarded. Some bound proteins, including lactic dehydrogenase, were eluted at 0.2 M KC1 added to the Tris buffer. Application of 1 M instead of 0.2 M KC1 decreased the subsequent yield of malic enzyme and did not improve the purity. Malic enzyme was eluted specifically by a linear gradient of NADP, and the recovery of activity was 51% of that present in the crude tissue extract (Table 1). The EDTA included in the buffers improved the recovery and the specific activity of the enzyme. The enzyme from the affinity chromatography column had a specific activity of 4.1 units/mg of protein at 25°C (Table 1) and did not contain detectable levels of glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, and NADPH oxidase. Glutathione reductase in the preparation oxidized NADPH at 9% of the rate that malic enzyme reduced NADP. Electrophoresis in polyacrylamide gels produced one band which stained for protein, and a corresponding band appeared in an identical gel stained for enzyme activity. When the electrophoresis was conducted in the presence
374
YEUNG
AND
CARRICO
of sodium dodecyl sulfate, five bands were evident in gels stained for protein. Gel filtration of the enzyme on Sephadex G-200 increased the specific activity 1.7-fold (Table 1) to give 7.0 and 26.2 units/mg of protein at 25 and 40°C respectively. One band was obtained by electrophoresis in both the presence and absence of sodium dodecyl sufate (Fig. 2). Glutathione reductase activity was 4% of the malic enzyme activity based on the relative rates of oxidation and reduction of NADP(H). The entire purification procedure, including preparation of the tissue extract, was completed in 50 hr. Inhibition of Malic Enzyme by N6-(&Aminohexyl)-Adenosine Bisphosphate
2 ‘,5 ‘-
The activity of malic enzyme in the presence of various levels of N6(6+uninohexyl)-adenosine 2’,5’-bisphosphate was measured at various concentrations of NADP. Reciprocal plots of the reaction rates indicated that the ligand inhibited competitively, and an inhibition constant of 50 PM N6-(6aminohexyl)-adenosine 2’,5’-bisphosphate was calculated. DISCUSSION
Published procedures for purification of NADP-dependent malic enzyme by conventional methods provide 10 to 30% recovery of enzyme activity and require 1 to 2 weeks of processing. By employing affinity chromatography, we isolated the enzyme from a crude chicken liver extract in 50 hr and over 40% yield (Table 1). Brodelius et al. (4) chromatographed a yeast extract on immobilized N6-(6aminohexyl)-adenosine 2’,5’-bisphosphate and isolated several NADP-dependent dehydrogenases by development of the chromatogram with a gradient of NADP. We have defined conditions for binding of malic enzyme to the immobilized ligand and have achieved greater than 200fold purification by elution with gradients of KC1 and NADP. Among the NADP-dependent enzymes which are known to bind to the ligand (4), only glutathione reductase was found in the preparation following affinity chromatography. Although results of gel electrophoresis and measurements of specific activity indicated that the enzyme preparation was not pure at this point, it was satisfactory for some purposes, such as use in conjunction with enzymic cycling (5). The enzyme was purified further by gel filtration and then one component was observed by gel electrophoresis (Fig. 2). Also, the specific activity, 26.2 units/mg of protein at 4O”C, was 94% of the value reported by Silpanta and Goodridge (2) for their preparation from chicken liver.
PURIFICATION
OF MALIC
ENZYME
375
REFERENCES 1. Hsu, R. Y., and Lardy, H. A. (1967) J. Biol. Chem. 242, 520. 2. Silpanta, P., and Goodridge, A. G. (1971) J. Biol. Chem. 246, 57.54. 3. Li, J. J., Ross, C. R., Tepperman, H. M., and Tepperman, J. (1975) J. Biol. Chem. 250, 141.
4. 5. 6. 7. 8.
Brodelius, P., Larsson, P-O., and Mosbach, K. (1974) Eur. J. Biochem. 47, 81. Kato, T., Berger, S. J., Carter, J. A., and Lowry, 0. H. (1973) Anal. Biochem. 53, 86. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anal. Biochem. 60, 149. Wise, E. M., Jr., and Ball, E. C. (1964) Proc. Nat. Acad. Sci. USA 52, 1255. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265. 9. Davis, B. J. (1%4) Ann. N. Y. Acad. Sci. 121, 404. 10. Henderson, N. S. (1966) Arch. Biochem. Biophys. 117, 28. 11. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244, 4406.
YEUNGANDROBERT
J. CARRICO
Ames Company, Division of Miles Laboratories, Elkhart, Indiana 46514
Inc.,
Received November 11, 1975; accepted April 20, 1976 A simple and rapid method for the purification of NADP-dependent malic enzyme from chicken liver is described. A crude tissue extract was chromatographed on an affinity column containing immobilized W-(6arninohexyl)adenosine 2’,5’-bisphosphate. Malic enzyme bound to the ligand and was eluted specifically by a gradient of NADP. Then the enzyme was purified further by gel filtration. The entire procedure requires 50 hr and provides the enzyme in about 40% yield. The purified enzyme migrated as one band during disc electrophoresis in polyacrylamide gel.
NADP-dependent malic enzymes have been isolated from several sources by methods which employ ethanol and ammonium sulfate fractionations, ion-exchange chromatography, and gel filtration (l-3). Although the crystalline enzyme has been prepared (l), the purification procedures are rather time consuming. Brodelius et al. (4) have shown that N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate inhibits several NADP-dependent enzymes, and these were purified by affinity chromatography on an immobilized form of the inhibitor. We have found that the nucleotide derivative also inhibits malic enzyme from chicken liver, and this enzyme can be purified rapidly by affinity chromatography. The purification procedure is of special interest because malic enzyme is used in recently described enzymic-cycling assay for NAD (5). MATERIALS
AND METHODS
The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO.): NAD, NADP, 6-chloropurine riboside, malic acid, glutathione (oxidized), dithiothreitol, phenazine methosulfate, nitroblue tetrazolium, 6-phosphogluconic acid. Lithium lactate, glucose 6-phosphate, NADPH, and a-ketoglutaric acid were purchased from Calbiochem (San Diego, Calif.). Sepharose4B and Sephadex G-200 were from Pharmacia Fine Chemicals (Piscataway, NJ). Glutamic acid and cyanogen bromide were products of Eastman Chemicals (Rochester, N.Y.). 369 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
370
YEUNG
AND
CARRICO
Fresh chicken liver was obtained from a local slaughter house and stored at -20°C until needed. Synthesis of N6-(6-aminohexyl)-adenosine 2’S’-bisphosphate. This synthesis was carried out according to the method of Brodelius et al. (4). The extinction coefficient reported by these authors was used to determine concentrations of the purified nucleotide. Binding of N6-(6-aminohexyl)-adenosine 2 ‘,5 ‘-bisphosphate to Sepharose-4B. One hundred milliliters of Sepharose-4B was activated with cyanogen bromide by the method of March et al. (6). The activated Sepharose was reacted with 455 pmol of the ligand in 100 ml of 0.2 M NaHCO,, pH 9.5, at 4°C for 18 hr. Then the liquid phase was separated by filtration on a sintered-glass funnel, and the optical absorbance at 265 nm was measured. The results indicate that 3.3 pmol of the ligand was bound per milliliter of Sepharose. Enzyme assays. Assays for enzyme activity were conducted at 25°C in 30 mM Tris-HCl buffer, pH 7.4, containing 1 mM dithiothreitol unless other conditions are indicated. Changes in absorbance at 340 nm due to reduction or oxidation of NAD(H) or NADP(H) were measured spectrophotometrically. The substrate and cofactor concentrations employed for the assays of glutamic dehydrogenase, glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase were the same as those used by Brodelius et al. (4). Assays for lactic dehydrogenase were conducted with 0.1 M lithium lactate and 10 mM NAD. Malic enzyme was assayed according to Wise and Ball (7) with some modifications. The reaction mixture contained 66 mM Tris-HCl, pH 7.4, 0.3 mM dithiothreitol, 4 mM MgC&, 0.2 mM NADP, and 0.5 mM malate. A unit of activity reduces 1 pmol of NADP/min. Studies of the inhibition of malic enzyme activity by N6-(6aminohexyl)adenosine 2’,5’-bisphosphate were conducted by measurement of the increase in fluorescence intensity due to reduction of NADP by 1 mM malate. The levels of NADPH and inhibitor were varied and the reaction was initiated by the addition of 0.2 unit of malic enzyme/ml. NADPH oxidase activity in malic enzyme preparations was measured fluorometrically. The reaction solution contained 30 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, and 3 PM NADPH. Protein assay. Measurements of protein were conducted according to the method of Lowry et al. (8), employing bovine serum albumin as the standard. Purification of NADP-dependent malic enzyme. Fifty grams of chicken liver and 150 ml of 30 mM Tris-HCl, pH 7.7, containing 0.25 M sucrose, 1 mM dithiothreitol, and 0.1 mM EDTA were homogenized in a Sorvall Omnimixer for 2 min at 0°C. Particulate material was separated by centrifugation at 30,OOOg for 30 min and then solid (NH&SO, was added
PURIFICATION
OF MALIC
ENZYME
371
to the supernatant to give 20% saturation. The suspension was centrifuged as above and the supernatant was brought to 70% saturation by slow addition of (NH&SO, over a period of 1 hr. The pellet obtained by centrifugation was dissolved in 150 ml of 30 mM Tris-HCI buffer, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. About 70 units of malic enzyme was applied to the affinity column. A 2.5 x 32-cm column of immobilized N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate was equilibrated at 5°C with 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. The crude malic enzyme was passed through the column at 2 ml/min, and then the column was washed with 200 ml of the Tris buffer. Next, the chromatogram was developed with 400 ml of the buffer containing 0.2 M KC1 followed by 300 ml of buffer without KCl. At this point, the absorbance of the effluent at 280 nm was less than 0.03 and a linear gradient of NADP, 0 to 0.5 mM (500 ml total), in 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA was applied at a flow rate of 2 mUmin. The effluent was collected in 6-ml fractions, and these were assayed for enzyme activity. Fractions containing malic enzyme were pooled as described below and concentrated to 4 to 6 ml by pressure dialysis. The concentrated enzyme preparation was applied to a 2.5 x 90-cm column of Sephadex G-200 which was previously equilibrated with 30 mM Tris-HCl, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA. The chromatogram was developed with the same buffer and malic enzyme eluted between 30 and 36% of the bed volume. The fractions with more than 0.1 unit of enzyme activity/ml were pooled and concentrated by pressure dialysis. To regenerate the affinity chromatography medium, it was stirred gently in 500 ml of 6 M urea containing 2 M KC1 and filtered on a sintered-glass funnel. Then it was washed with 350 ml of 30 mM Tris-HCl buffer, pH 7.7, containing 1 mM dithiothreitol and 0.1 mrvr EDTA and repacked into a column. The immobilized ligand was used for 10 preparations of malic enzyme without notable change in binding capacity. Disc efectrophoresis. Electrophoresis in 5% polyacrylamide gels was conducted according to the method of Davis (9). Gels were stained for protein with amido black and for malic enzyme activity by the method of Henderson (10). Electrophoresis in the presence of sodium dodecyl sulfate was carried out in 5% polyacrylamide gels by the method of Weber and Osborn (11). RESULTS Purification
of Malic Enzyme
In preliminary studies we subjected a crude chicken liver extract to affinity chromatography after centrifugation at 78,SOOg for 1 hr. The
372
YEUNG AND CARRICO TABLE SPECIFIC
ACITIVITY
AND RECOVERY
1
OF MALIC
ENZYME
DURING
PURIFICATION
Preparation assayed
Enzyme activity (units)
Specific activity (units/mg of protein
Recovery (%I
Liver extract Ammonium sulfate Affinity chromatography Gel filtration
79 70 41 33
0.017 0.019 4.1 7.0
100 88 51 41
chromatography was completed successfully; however, lipid-like material accumulated at the top of the column and reduced the flow rate. The interfering material was separated from the malic enzyme by ammonium sulfate fractionation. Over 80% of the enzyme activity was recovered in the precipitate formed between 20 and 70% saturation with ammonium sulfate (Table 1). A typical elution profile obtained by chromatography of the ammonium sulfate fraction on the affinity column is shown in Fig. 1. Most proteins
l.zj
g 0.6 %04 P 0.2
i 0.6 L0 200 EFFLUENT
VOLUME (ml)
1. Elution profile obtained by chromatography of a crude chicken liver extract on immobilized NB-(6-aminohexyl)-adenosine 2’,5’-bisphosphate. A chicken liver extract was fractionated with ammonium sulfate as described in Materials and Methods. The material precipitated by 70% ammonium sulfate was dissolved in 150 ml of 30 mM Tris-HCI, pH 7.7, containing 1 mM dithiothreitol and 0.1 mM EDTA and passed at a flow rate of 2 mUmin into a 2.5 x 32-cm column of Np-(6aminohexyl)-adenosine 2’,5’-bisphosphate bound to Sepharose. At (A) the column was washed with the Tris-HCl buffer, containing 1 mrvt dithiothreitol and 0.1 mM EDTA, and at (B) 0.2 M KC1 was included in the buffer. Then the KC1 was washed from the column with the buffer (C), and finally a linear gradient of NADP was applied at (D). Six-milliliter fractions were collected, and the absorbance at 280 nm (0) was measured in fractions preceding the NADP gradient. The elution profiles for lactic dehydrogenase (0) and malic enzyme (A) are shown. The broken line indicates that NADP concentration. The portion of the chromatogram pooled for the purified malic enzyme preparation is indicated by the horizontal line. FIG.
PURIFICATION
OF MALIC
ENZYME
373
FIG. 2. Disc electrophoresis of purified malic enzyme. Purified malic enzyme was examined by electrophoresis in polyacrylamide gels as described in Materials and Methods. About 20 pg of protein was applied to each gel. Gel (A) was stained for protein with amido black, and gel (B) was stained for malic enzyme activity. Electrophoresis for gel (C) was conducted in the presence of sodium dodecyl sulfate, and the gel was stained with Coomassie blue.
passed through the column unretarded. Some bound proteins, including lactic dehydrogenase, were eluted at 0.2 M KC1 added to the Tris buffer. Application of 1 M instead of 0.2 M KC1 decreased the subsequent yield of malic enzyme and did not improve the purity. Malic enzyme was eluted specifically by a linear gradient of NADP, and the recovery of activity was 51% of that present in the crude tissue extract (Table 1). The EDTA included in the buffers improved the recovery and the specific activity of the enzyme. The enzyme from the affinity chromatography column had a specific activity of 4.1 units/mg of protein at 25°C (Table 1) and did not contain detectable levels of glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, and NADPH oxidase. Glutathione reductase in the preparation oxidized NADPH at 9% of the rate that malic enzyme reduced NADP. Electrophoresis in polyacrylamide gels produced one band which stained for protein, and a corresponding band appeared in an identical gel stained for enzyme activity. When the electrophoresis was conducted in the presence
374
YEUNG
AND
CARRICO
of sodium dodecyl sulfate, five bands were evident in gels stained for protein. Gel filtration of the enzyme on Sephadex G-200 increased the specific activity 1.7-fold (Table 1) to give 7.0 and 26.2 units/mg of protein at 25 and 40°C respectively. One band was obtained by electrophoresis in both the presence and absence of sodium dodecyl sufate (Fig. 2). Glutathione reductase activity was 4% of the malic enzyme activity based on the relative rates of oxidation and reduction of NADP(H). The entire purification procedure, including preparation of the tissue extract, was completed in 50 hr. Inhibition of Malic Enzyme by N6-(&Aminohexyl)-Adenosine Bisphosphate
2 ‘,5 ‘-
The activity of malic enzyme in the presence of various levels of N6(6+uninohexyl)-adenosine 2’,5’-bisphosphate was measured at various concentrations of NADP. Reciprocal plots of the reaction rates indicated that the ligand inhibited competitively, and an inhibition constant of 50 PM N6-(6aminohexyl)-adenosine 2’,5’-bisphosphate was calculated. DISCUSSION
Published procedures for purification of NADP-dependent malic enzyme by conventional methods provide 10 to 30% recovery of enzyme activity and require 1 to 2 weeks of processing. By employing affinity chromatography, we isolated the enzyme from a crude chicken liver extract in 50 hr and over 40% yield (Table 1). Brodelius et al. (4) chromatographed a yeast extract on immobilized N6-(6aminohexyl)-adenosine 2’,5’-bisphosphate and isolated several NADP-dependent dehydrogenases by development of the chromatogram with a gradient of NADP. We have defined conditions for binding of malic enzyme to the immobilized ligand and have achieved greater than 200fold purification by elution with gradients of KC1 and NADP. Among the NADP-dependent enzymes which are known to bind to the ligand (4), only glutathione reductase was found in the preparation following affinity chromatography. Although results of gel electrophoresis and measurements of specific activity indicated that the enzyme preparation was not pure at this point, it was satisfactory for some purposes, such as use in conjunction with enzymic cycling (5). The enzyme was purified further by gel filtration and then one component was observed by gel electrophoresis (Fig. 2). Also, the specific activity, 26.2 units/mg of protein at 4O”C, was 94% of the value reported by Silpanta and Goodridge (2) for their preparation from chicken liver.
PURIFICATION
OF MALIC
ENZYME
375
REFERENCES 1. Hsu, R. Y., and Lardy, H. A. (1967) J. Biol. Chem. 242, 520. 2. Silpanta, P., and Goodridge, A. G. (1971) J. Biol. Chem. 246, 57.54. 3. Li, J. J., Ross, C. R., Tepperman, H. M., and Tepperman, J. (1975) J. Biol. Chem. 250, 141.
4. 5. 6. 7. 8.
Brodelius, P., Larsson, P-O., and Mosbach, K. (1974) Eur. J. Biochem. 47, 81. Kato, T., Berger, S. J., Carter, J. A., and Lowry, 0. H. (1973) Anal. Biochem. 53, 86. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anal. Biochem. 60, 149. Wise, E. M., Jr., and Ball, E. C. (1964) Proc. Nat. Acad. Sci. USA 52, 1255. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265. 9. Davis, B. J. (1%4) Ann. N. Y. Acad. Sci. 121, 404. 10. Henderson, N. S. (1966) Arch. Biochem. Biophys. 117, 28. 11. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244, 4406.