Purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 74, No. 4, pp , April 1977 Biochemistry Purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver (affinity chromatography/active and inactive enzyme/immunodiffusion/polyacrylamide gel electrophoresis) DON A. KLEINSEK, S. RANGANATHAN, AND JOHN W. PORTER Lipid Metabolism Laboratory, Veterans Administration Hospital, and the Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin Communicated by David E. Green, January 14,1977 ABSTRACT A procedure for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC solubilized from rat liver microsomes is reported. This enzyme has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This represents a 4100 fold purification over the activity in microsomes, and a specific activity that is approximately 20-fold greater than the highest previously reported value. The enzyme is judged to be homogeneous on the basis of sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, polyacrylamide disc gel electrophoresis, and immunoanalysis. Data are also presented that indicate the separation of enzymatically active and inactive species of 3-hydroxy-3- methylglutaryl-coenzyme A reductase on affinity chromatography on a coenzyme A column. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:nadp+ oxidoreductase (CoA-acylating); EC ] catalyzes the reduction of HMG-CoA to mevalonic acid, the rate-limiting step of cholesterol biosynthesis in liver (1-3). Therefore, a number of researchers have focused their attention on the regulation of this enzyme. However, to study the modulation of HMG-CoA reductase under various physiological states a method is required to quantitate the amount of enzyme present. This can be achieved by either direct isolation of the enzyme by a reproducible purification procedure or by immunoprecipitation using monospecific antiserum to the enzyme. A number of procedures have been reported for the solubilization and partial purification of HMG-CoA reductase from the microsomal membrane (4-8). In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9-11). However, the enzyme activities of these preparations were very low ( nmol of mevalonate formed per min/mg of protein). In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein. In this paper we report the purification of HMG-CoA reductase from rat liver by a combination of standard protein fractionation steps and coenzyme A affinity chromatography. This preparation also has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This value is approximately 20-fold greater than the best value previously reported. As a part of this study we also show that enzymatically active and inactive species of HMG-CoA reductase are separated by affinity chromatography. This separation suggests the possibility that cholesterol synthesis may be regulated in vvo by the interconversion of these species. Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methylglutarylcoenzyme A reductase; NaDodSO4, sodium dodecyl sulfate MATERIALS AND METHODS Materials. Chemicals were obtained from the following sources: 3-hydroxy-3-methyl[3-'4C]glutaric acid, New England Nuclear; coenzyme A, thioester-linked agarose-hexane-coenzyme A, and dithiothreitol, P-L Biochemicals, Inc.; glucose- 6-phosphate, Nutritional Biochemicals Corp.; 3-hydroxy-3- methylglutaric acid, glucose-6-phosphate dehydrogenase, NADP+, and NADPH, Sigma Chemical Co.; Bio-Gel, Bio-Rad Laboratories; and cholestyramine, Mead Johnson Laboratories. All other chemicals and reagents used were of analytical grade. Treatment of Animals. Male albino Holtzman rats weighing g were used for all experiments. The animals were housed in a light-controlled room in which the dark period was maintained from 1200 to 2400 hours. The animals were fed ad lib. a 2% cholestyramine Wayne Lab Blox powdered diet for a minimum of 4 days to effect maximum liver HMG-CoA reductase activity. Preparation of Microsomes. The animals were sacrificed by decapitation at 1800 hours, the diurnal high point of HMG-CoA reductase activity. The livers were excised and immediately placed in ice-cold homogenization medium which contained 50 mm potassium phosphate buffer (ph 7.0)/0.2 M sucrose/2 mm dithiothreitol (buffer I). Livers were homogenized in this medium (2 ml/g of liver) in a Waring blendor for 15 sec, followed by three strokes with a motor-driven Teflon pestle in a Potter-Elvehjem type glass homogenizer. The homogenate was centrifuged for 10 min at 15,000 X g and the supernatant solution was centrifuged at 100,000 X g for 75 min. The microsomal pellet was resuspended in buffer I containing 50 mm EDTA and recentrifuged at 100,000 X g for 60 min. This pellet was used for isolation of the enzyme. All of the above operations were carried out at 4. Solubilization of Enzyme. The method of Heller and Gould (7) was used to solubilize the enzyme. Microsomal pellets were frozen at -20 for at least 2 hr. After thawing at room temperature the microsomes were homogenized in solubilization buffer that contained 50 mm potassium phosphate (ph 7.0)/0.1 M sucrose/2 mm dithiothreitol/50 mm KCI/30 mm EDTA. A Potter-Elvehjem homogenizer with a tight-fitting Teflon pestle was used. After standing for 15 min at room temperature, the suspension was centrifuged at 100,000 X g for 60 min at 200. The supernatant solution was collected and used for the purification of the enzyme. All further operations were carried out at room temperature. Assay Systems. Two assay systems were used to measure HMG-CoA reductase activity. For measuring microsomal enzyme activity, an adaptation of the NADPH-generating radiochemical method previously described (13) was used. The 0.5-ml reaction volume contained potassium phosphate buffer (ph 7.0), 50,mol/dithiothreitol, 2,umol/glucose-6-phosphate,

2 1432 Biochemistry: Kleinsek et al. Table 1. Purification of HMG-CoA reductase from rat liver microsomes Specific Total activity Purification protein Total (units/mg Purifistep (mg) units protein) Yield cation Microsomal suspension 4,800 11, Soluble extract 198 3, (NH4)2SO4, 35-50% 65 3, Heat treatment 13 2, (NH4)2SO4, 0-50% 9 2, Bio-Gel filtration* , CoA affinity column , ,078 The data of this table were obtained during the purification of HMG-CoA reductase from 25 rat livers. * Sucrose density gradient centrifugation may be substituted for the Bio-Gel filtration step. This procedure was used in the preparation of material for immunodiffusion analysis. Proc. Nati. Acad. Sci. USA 74 (1977) NADPH oxidation was determined from the change in absorbance at 340 nm using a glass cuvette with a 2-mm light path. This reaction results in 1 nmol of mevalonate formed per 2 nmol of NADPH oxidized. One unit of HMG-CoA reductase activity is defined as the quantity of enzyme that produces 1 nmol of mevalonate in 1 min at 37'. Protein determinations were carried out by the method of Lowry et al. (14). Polyacrylamide Gel Electrophoresis. The procedure of Weber and Osborn (15) was used for NaDodSO4/polyacrylamide gel electrophoresis. NaDodSO4/5% polyacrylamide gels were fixed and the NaDodSO4 was leached out in 20% sulfosalicyclic acid (16) prior to staining with 0.25% Coomassie brilliant blue. Polyacrylamide disc gels (5%, ph 8.9) were made with 30% glycerol. The electrophoresis was carried out in 5 mm Tris/35 mm glycine at ph 8.3. Antisera and Immunodiffusion. Crude antisera was obtained by subcutaneously injecting a 5-kg rabbit at 2-week intervals with 1 mg of supernatant protein from the heat-treatment step. Blood was withdrawn after 4, 6, and 8 weeks of the initial injection and allowed to clot; the serum was concentrated with a 0-50% (NH4)2SO4 fractionation. The antisera pellet was dissolved in a 10 mm potassium phosphate (ph 7.0)/0.9% NaCl solution and stored at -20. Immunodiffusion slides consisted of a 0.5% agar/0.9% NaCl matrix. 2,umol/NADP+, 0.5 Mmol/DL-[3-14C]HMG-CoA, 0.15,gmol/ glucose-6-phosphate dehydrogenase, 1.25 units/enzyme protein, ,tg. The reaction was carried out at 370 for 5-10 min and terminated by the addition of 50 Al of 2.4 M HCL. A 200-IdI aliquot of the incubation mixture was spotted directly onto activated Silica Gel G thin-layer plates and the chromatogram was developed in benzene/acetone (1:1, vol/vol). HMG-CoA reductase activity in the solubilized fractions was assayed spectrophotometrically. The reaction volume was 0.5 ml and it consisted of potassium phosphate buffer (ph 7.0), 50,umol/dithiothreitol, 2,Mmol/NADPH, 0.30,tmol/DL-HMG- CoA, 0.15,gmol/enzyme protein, ,gg. The reaction mixture, without substrate, was first incubated for 5 min at 370 and the assay was carried out at 370 in a Gilford 2400S spectrophotometer after addition of HMG-CoA. The rate of 0.5 M KCI V I ~~~~~~~~~~0 0I E It c 0.30-' 600 z 0 I 0.20-I zi 0 m-j FRACTION NUMBER 8 10 U- 400 a: 0j 200u FIG. 1. Affinity chromatography of HMG-CoA reductase on a coenzyme A column. The enzyme was added to the column, 0.2 ml of wet gel packed in a pasteur pipette, at 40 in 25 mm potassium phosphate buffer (ph 7.0)/1 mm dithiothreitol/1 mm EDTA/10% sucrose. The flow rate was 1 ml/10 min. The unbound protein fraction was concentrated and saved for immunoanalysis. HMG-CoA reductase was eluted by increasing the ionic strength of the buffer by addition of KCl to a concentration of 0.5 M. The elution flow rate was 1 ml/min and fractions of 1 ml were collected. 2 N z Li RESULTS Purification of Enzyme. HMG-CoA reductase has been purified to near homogeneity from a solubilized extract of rat liver microsomes. A typical purification is presented in Table 1. After the solubilization of HMG-CoA reductase from microsomes, the soluble extract was subjected to a 35-50% saturation with (NH4)2SO4. This resulted in a 2- to 3-fold purification and a small loss of enzyme activity. The protein pellet was dissolved in buffer containing 50 mm potassium phosphate (ph 7.0)/3 mm dithiothreitol/30% (vol/vol) glycerol/1.0 M KCI at a concentration of 6-8 mg/ml. The enzyme solution was heated at 65 for 6 min and denatured protein was'removed by centrifugation at 100,000 X g for 30 min. The supernatant solution contained 70-80% of the starting enzyme activity and an increase of 3- to 4-fold in specific activity of the enzyme was observed. The heat-treated extract was then concentrated, after diluting 1:1 with solubilization buffer, by precipitation of protein by 0-50% saturation with (NH4)2SO4. The protein pellet was dissolved in a minimal volume of 50 mm potassium phosphate (ph 7.0)/2 mm dithiothreitol/30 mm EDTA/50 mm KCI/10% sucrose. The enzyme solution was loaded onto a Bio-Gel A-0.5m gel filtration column (2 X 44 cm). The peak fractions of enzyme activity in the eluant were'purified 8-fold in enzyme activity with a 38% recovery of enzyme protein. The fractions eluted from the gel were concentrated and then dialyzed in collodion bags. The dialyzed protein was applied to a thioester-linked agarose/hexane/coenzyme A column. Details of the affinity binding and elution conditions are reported in the legend to Fig.' 1. The affinity step routinely resulted in a % recovery of enzyme activity with a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. Criteria for Homogeneity of Enzyme. One major staining band was obtained when 40 jig of purified enzyme was electrophoresed on NaDodSO4/polyacrylamide disc gel (Fig. 2). Similarly, only a single band was observed when 10-20,ug of purified enzyme was electrophoresed on 5% polyacrylamide disc gel (Fig. 3). A third criterion of homogeneity of the enzyme is shown in Fig. 4. A single immunoprecipitin band was ob-

3 Biochemistry: Kleinsek et al. Proc. Natl. Acad. Sci. USA 74 (1977) 1433 FIG. 2. NaDodSO4 disc gel electrophoresis of HMG-CoA reductase. Protein, 40 Mug of affinity-purified enzyme, was electrophoresed in 0.1% NaDodSO4 on a 5% polyacrylamide disc gel. An RF of approximately 0.65 was obtained for the single staining band. tained when purified enzyme was reacted against crude antisera. Evidence for Active and Inactive Enzyme Species. The protein fraction that did not bind to the coenzyme A affinity gel contained a species that was immunologically identical to the protein fraction that bound to, and was eluted off, the affinity column. This is demonstrated by the partial identity of antigenic determinants in well c with those in wells b and d. Because the enzyme solution had been centrifuged at 100,000 X g prior to affinity chromatography the enzymatically inactive species that did not bind to the affinity column was not denatured protein. In addition, the protein fraction not bound to the affinity column was not inactivated because of cold lability (17) because this fraction showed no reactivation of catalytic properties after preincubation at 37. a b FIG. 3. Polyacrylamide disc gel electrophoresis of HMG-CoA reductase. Protein, 10,gg (a) and 20,gg (b), was stained with 0.25% Coomassie brilliant blue. Five percent or less of active HMG-CoA reductase did not bind to the column, and this protein was recovered in the unbound eluant. However, this amount of protein did not account for the line of confluence between unbound and bound fractions on immunoanalysis. Furthermore, the staining profiles of the two fractions on NaDodSO4/polyacrylamide gels (Figs. 2 and 5) indicated that these two species were identical in their migration characteristics. Hence it is concluded that the majority of the protein unbound on affinity chromatography was inactive HMG-CoA reductase. DISCUSSION The procedure for the purification of HMG-CoA reductase reported in this paper yields a protein with a specific activity

4 1434 Biochemistry: Kleinsek et al. Proc. Nati. Acad. Sci. USA 74 (1977) FIG. 4. Ouchterlony double-diffusion precipitation of affinity protein fractions. The center well is antisera prepared against the heat-treated enzyme preparation. Wells a and c contain 1.5,ug and 7 jg, respectively, of affinity-bound enzyme. Wells b and d represent 8 jug and 16 gg, respectively, of the protein fraction that did not bind to the affinity column. of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. Polyacrylamide disc gel and NaDodSO4/polyacrylamide disc gel electrophoresis of the purified enzyme show only a single major staining band. In addition, immunodiffusion of the enzyme against antisera to crude enzyme yields only one precipitin band. These results are evidence for homogeneity of our preparation. Prior studies from three other laboratories have reported the preparation of HMG-CoA reductase from rat liver. However, each of these preparations had an activity that was 5% or less of the activity we report for the rat liver and yeast enzymes. In the initial reported preparation, Higgins et al. (9) solubilized microsomal-bound HMG-CoA reductase with sodium deoxycholate and then purified the enzyme to an activity of 10 nmol of mevalonate formed per min/mg of protein. More recently, Heller and Shrewsbury (11) and Tormanen et al. (10) reported the isolation of HMG-CoA reductase. Each of these preparations was solubilized from microsomes by a slow freeze-thaw technique and then purified by different procedures. Heller and Shrewsbury (11) reported a specific activity of 462 nmol of' mevalonate formed per min/mg of protein, while Tormanen et al. (10) obtained a specific activity of 516. Obviously, the HMG-CoA reductase preparations reported previous to this paper were (i) impure or (ii) a mixture of active and inactive enzyme or (iii) they contained significant amounts of an inhibitor (possibly cholesterol) bound to the enzyme, inasmuch as the best of these systems had an activity of 5% or less of the value we report. A partial explanation for the discrepancy has been supplied by the data in this paper. Affinity chromatography on a coenzyme A column separated HMG-CoA reductase into active and inactive species. The fraction that passed through the affinity column contained little or no HMG-CoA reductase activity. However, this fraction gives an immunoprecipitin band that is continuous with that given by the active enzyme bound to the coenzyme A affinity column. Furthermore, when both of these species were subjected to FIG. 5. NaDodSO4 disc gel electrophoresis of the protein (30,g) not bound to a CoA affinity gel column. An RF of approximately 0.65 was obtained for the major staining band. NaDodSO4 gel electrophoresis, identical migratory behaviors of the proteins were noted. The separation of active and inactive species of HMG-CoA reductase is a confirmation and an extension of previous studies (18-21) that indicated that HMG-CoA reductase activity of crude liver preparations could be increased or decreased by incubation with appropriate subeellular fractions. Presumably these studies involved a change in enzyme activity through covalent modification of the enzyme. Hence it will be of interest to learn from future work the difference between active and inactive HMG-CoA reductase species and the mechanism of regulation of their interconversion. This investigation was supported in part by grants from the Wisconsin Heart Association and the National Heart and Lung Institute (no. HL-16364) of the National Institutes of Health, U.S. Public Health Service. 1. Siperstein, M. D. & Fagan, V. M. (1966) J. Biol. Chem. 241, White, L. W. & Rudney, H. (1970) Biochemistry 9, Shapiro, D. & Rodwell, V. W. (1972) Biochemistry 11, Linn, T. C. (1967) J. Biol. Chem. 242, Kawachi, T. & Rudney, H. (1970) Biochemistry 9, Brown, M. S., Dana, S. E., Dietschy, J. M. & Siperstein, M. D. (1973) J. Biol. Chem. 248, Heller, R. A. & Gould, R. G. (1973) Biochem. Blophys. Res. Commun. 50,

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