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Purification of 3-Hydroxy-3-Methylglutaryl

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Purification of 3-Hydroxy-3-Methylglutaryl Proc. Nati. Acad. Sci. USA Vol. 74, No. 4, pp. 1431-1435, 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 53706 Communicated by David E. Green, January 14,1977 ABSTRACT A procedure for the purification of 3-hy- MATERIALS AND METHODS droxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341 solubilized Materials. Chemicals were obtained from the following from rat liver microsomes is reported. This enzyme has a spe- sources: 3-hydroxy-3-methyl[3-'4C]glutaric acid, New England cific activity of 9,000-10,000 nmol of mevalonate formed per Nuclear; coenzyme A, thioester-linked agarose-hexane-coen- min/mg of protein. This represents a 4100 fold purification over the activity in microsomes, and a specific activity that is ap- zyme A, and dithiothreitol, P-L Biochemicals, Inc.; glucose- proximately 20-fold greater than the highest previously reported 6-phosphate, Nutritional Biochemicals Corp.; 3-hydroxy-3- value. The enzyme is judged to be homogeneous on the basis methylglutaric acid, glucose-6-phosphate dehydrogenase, of sodium dodecyl sulfate/polyacrylamide disc gel electro- NADP+, and NADPH, Sigma Chemical Co.; Bio-Gel, Bio-Rad phoresis, polyacrylamide disc gel electrophoresis, and immu- Laboratories; and cholestyramine, Mead Johnson Laboratories. noanalysis. Data are also presented that indicate the separation All other chemicals and reagents used were of analytical of enzymatically active and inactive species of 3-hydroxy-3- methylglutaryl-coenzyme A reductase on affinity chromatog- grade. on a A column. Treatment of Animals. Male albino Holtzman rats weighing raphy coenzyme 180-200 g were used for all experiments. The animals were 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) re- housed in a light-controlled room in which the dark period was ductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); maintained from 1200 to 2400 hours. The animals were fed ad EC 1.1.1.34] catalyzes the reduction of HMG-CoA to mevalonic lib. a 2% cholestyramine Wayne Lab Blox powdered diet for acid, the rate-limiting step of cholesterol biosynthesis in liver a minimum of 4 days to effect maximum liver HMG-CoA re- (1-3). Therefore, a number of researchers have focused their ductase activity. attention on the regulation of this enzyme. However, to study Preparation of Microsomes. The animals were sacrificed the modulation of HMG-CoA reductase under various physi- by decapitation at 1800 hours, the diurnal high point of ological states a method is required to quantitate the amount HMG-CoA reductase activity. The livers were excised and of enzyme present. This can be achieved by either direct iso- immediately placed in ice-cold homogenization medium which lation of the enzyme by a reproducible purification procedure contained 50 mM potassium phosphate buffer (pH 7.0)/0.2 M or by immunoprecipitation using monospecific antiserum to sucrose/2 mM dithiothreitol (buffer I). Livers were homoge- the enzyme. nized in this medium (2 ml/g of liver) in a Waring blendor for A number of procedures have been reported for the solubi- 15 sec, followed by three strokes with a motor-driven Teflon lization and partial purification of HMG-CoA reductase from pestle in a Potter-Elvehjem type glass homogenizer. The ho- the microsomal membrane (4-8). In addition, workers in three mogenate was centrifuged for 10 min at 15,000 X g and the laboratories have reported the preparation of enzyme that yields supernatant solution was centrifuged at 100,000 X g for 75 min. only one band on immunodiffusion or sodium dodecyl sulfate The microsomal pellet was resuspended in buffer I containing (NaDodSO4) disc gel electrophoresis (9-11). However, the 50 mM EDTA and recentrifuged at 100,000 X g for 60 min. enzyme activities of these preparations were very low (10-516 This pellet was used for isolation of the enzyme. All of the above nmol of mevalonate formed per min/mg of protein). operations were carried out at 4°. In a previous study (12) we succeeded in purifying yeast Solubilization of Enzyme. The method of Heller and Gould HMG-CoA reductase to homogeneity. This enzyme had a (7) was used to solubilize the enzyme. Microsomal pellets were specific activity of approximately 10,000 nmol of mevalonate frozen at -20° for at least 2 hr. After thawing at room tem- formed per min/mg of protein. In this paper we report the perature the microsomes were homogenized in solubilization purification of HMG-CoA reductase from rat liver by a com- buffer that contained 50 mM potassium phosphate (pH 7.0)/0.1 bination of standard protein fractionation steps and coenzyme M sucrose/2 mM dithiothreitol/50 mM KCI/30 mM EDTA. A affinity chromatography. This preparation also has a specific A Potter-Elvehjem homogenizer with a tight-fitting Teflon activity of 9,000-10,000 nmol of mevalonate formed per pestle was used. After standing for 15 min at room temperature, min/mg of protein. This value is approximately 20-fold greater the suspension was centrifuged at 100,000 X g for 60 min at 200. than the best value previously reported. The supernatant solution was collected and used for the puri- As a part of this study we also show that enzymatically active fication of the enzyme. All further operations were carried out and inactive species of HMG-CoA reductase are separated by at room temperature. affinity chromatography. This separation suggests the possibility Assay Systems. Two assay systems were used to measure that cholesterol synthesis may be regulated in vvo by the in- HMG-CoA reductase activity. For measuring microsomal en- terconversion of these species. zyme activity, an adaptation of the NADPH-generating ra- diochemical method previously described (13) was used. The Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methylglutaryl- 0.5-ml reaction volume contained potassium phosphate buffer coenzyme A reductase; NaDodSO4, sodium dodecyl sulfate. (pH 7.0), 50,mol/dithiothreitol, 2,umol/glucose-6-phosphate, 1431 Downloaded by guest on September 24, 2021 1432 Biochemistry: Kleinsek et al. Proc. Nati. Acad. Sci. USA 74 (1977) Table 1. Purification of HMG-CoA reductase NADPH oxidation was determined from the change in ab- from rat liver microsomes sorbance 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 Specific of NADPH oxidized. One unit of HMG-CoA reductase activity Total activity is defined as the quantity of enzyme that produces 1 nmol of Purification protein Total (units/mg Purifi- mevalonate in 1 min at 37'. Protein determinations were car- step (mg) units protein) Yield cation ried out by the method of Lowry et al. (14). Microsomal Polyacrylamide Gel Electrophoresis. The procedure of suspension 4,800 11,255 2.3 100 1 Weber and Osborn (15) was used for NaDodSO4/polyacryl- Soluble extract 198 3,527 17.8 31 8 amide gel electrophoresis. NaDodSO4/5% polyacrylamide gels (NH4)2SO4, were fixed and the NaDodSO4 was leached out in 20% sulfo- 35-50% 65 3,189 49 28 22 salicyclic acid (16) prior to staining with 0.25% Coomassie Heat treatment 13 2,506 193 22 82 brilliant blue. Polyacrylamide disc gels (5%, pH 8.9) were made (NH4)2SO4, with 30% glycerol. The electrophoresis was carried out in 5 mM 0-50% 9 2,050 228 18 97 Tris/35 mM glycine at pH 8.3. Bio-Gel Antisera and Immunodiffusion. Crude antisera was ob- filtration* 0.42 777 1,850 7 787 tained by subcutaneously injecting a 5-kg rabbit at 2-week in- CoA affinity tervals with 1 mg of supernatant protein from the heat-treat- column 0.036 345 9,583 3 4,078 ment step. Blood was withdrawn after 4, 6, and 8 weeks of the initial injection and allowed to serum The data of this table were obtained during the purification of clot; the was concentrated HMG-CoA reductase from 25 rat livers. with a 0-50% (NH4)2SO4 fractionation. The antisera pellet was * Sucrose density gradient centrifugation may be substituted for the dissolved in a 10 mM potassium phosphate (pH 7.0)/0.9% NaCl Bio-Gel filtration step. This procedure was used in the preparation solution and stored at -20°. Immunodiffusion slides consisted of material for immunodiffusion analysis. of a 0.5% agar/0.9% NaCl matrix. 2,umol/NADP+, 0.5 Mmol/DL-[3-14C]HMG-CoA, 0.15,gmol/ RESULTS glucose-6-phosphate dehydrogenase, 1.25 units/enzyme pro- tein, 300-1200,tg. The reaction was carried out at 370 for 5-10 Purification of Enzyme. HMG-CoA reductase has been min and terminated by the addition of 50 Al of 2.4 M HCL. A purified to near homogeneity from a solubilized extract of rat 200-IdI aliquot of the incubation mixture was spotted directly liver microsomes. A typical purification is presented in Table onto activated Silica Gel G thin-layer plates and the chro- 1. After the solubilization of HMG-CoA reductase from mi- matogram was developed in benzene/acetone (1:1, vol/vol). crosomes, the soluble extract was subjected to a 35-50% satu- HMG-CoA reductase activity in the solubilized fractions was ration with (NH4)2SO4. This resulted in a 2- to 3-fold purifi- assayed spectrophotometrically. The reaction volume was 0.5 cation and a small loss of enzyme activity. The protein pellet ml and it consisted of potassium phosphate buffer (pH 7.0), 50 was dissolved in buffer containing 50 mM potassium phosphate ,umol/dithiothreitol, 2,Mmol/NADPH, 0.30,tmol/DL-HMG- (pH 7.0)/3 mM dithiothreitol/30% (vol/vol) glycerol/1.0 M CoA, 0.15,gmol/enzyme protein, 0.2-400,gg.
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