ARCHIVES OF BIOCHEMISTRY Vol. 197, No. 2, October 15,
AND BIOPHYSICS
pp. 493-499, 1979
Purification GENE
of 3-Hydroxy3-Methylglutaryl Reductase from Rat Liver
C. NESS, Department
CHRISTY
of Biochemistry,
D. SPINDLER, College Tampa,
Coenzyme
AND MARY
of Medicine, University Florida 33612
A
H. MOFFLER
of South
Florida,
Received February 22, 1979; revised June 7, 1979 A procedure for the purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.341 from rat liver microsomes has been developed. The enzyme preparations obtained by this procedure have specific activities of 16 to 23 pmol of mevalonate formed per minute per milligram of protein. These enzyme preparations were judged to be homogeneous on the basis of comigration of enzyme activity and protein on polyacrylamide gels.
The microsomal enzyme, 3-hydroxy-3methylglutaryl coenzyme A reductase, catalyzes the rate-limiting reaction in cholesterol biosynthesis (1). In order to gain a better understanding of the physiological mechanism(s) which regulate this enzyme, considerable effort has been devoted to purifying the reductase. Several reports have appeared (2-8) which describe procedures for obtaining homogeneous preparations of the reductase from rat liver. These procedures yield preparations with widely different specific activities and properties. Recently, it was shown that two of these previously reported homogeneous enzyme preparations (4, 5) were actually quite impure when analyzed by electrophoresis on polyacrylamide gels and assayed for enzyme activity as well as stained for protein (9). In this report we describe a purification procedure which sequentially utilizes Affi-Gel Blue and HMG-CoA agarose’
affinity chromatography. This procedure yields homogeneous enzyme preparations with specific activities of 16 to 23 pmol of mevalonate formed per minute per-milligram of protein. EXPERIMENTAL
PROCEDURE
Materials. RS-3-[3-14C-glutaryl]hydroxy-3-methylglutaryl coenzyme A was purchased from New England Nuclear. Agarose-hexane 3-hydroxy-3-methylglutaryl coenzyme A, Type 5 (HMG-CoA agarose) was purchased from P. L. Biochemicals. Affi-Gel Blue, 75-150 pm, Bio-Rad protein assay dye reagent concentrate, BioPhore 7.5% gels and AG l-x8 formate, 200-400 mesh were purchased from Bio-Rad Laboratories. The liquid scintillation cocktail, ACS, was purchased from Amersham. Cholestyramine (Questran) was purchased from Mead Johnson. Glucose 6-phosphate dehydrogenase (type XII), NADP+, NADPH, dithiothreitol, and unlabeled HMG-CoA were purchased from Sigma Chemical Company. Animals. Male Sprague-Dawley rats weighing 125-150 g were purchased from ARSiSpragueDawley, Madison, Wisconsin. The animals were housed in a light-controlled room in which the dark ’ Abbreviations used: HMG-CoA agarose, agaroseperiod was from 1000 to 2200. The animals were fed hexane 3-hydroxy-3-methylglutaryl coenzyme A; ground Wayne Lab Blox containing2% cholestyramine PESK buffer, 40 mM potassium phosphate, 30 mM for 6 to 8 days prior to being killed. Under these EDTA, 0.1 M sucrose, 50 mM potassium chloride, and conditions maximal HMG-CoA reductase activity oc1 mM dithiothreitol; solution A, 28% (w/v) a&amide curs at 3 h into the dark period which is referred to and 0.56% (w/v) N,N’-methylene-bisacrylamide; solu- as D-3. tion, B, 6.05% (w/v) Tris, 0.2% (v/v) N,N,N’,N’Preparation of microsomes. Cholestyramine-fed tetramethylethylenediamine (TEMED); solution C, rats were killed by decapitation at D-3 and their livers 0.14% (w/v) ammonium persulfate in 40% glycerol; were quickly removed and placed in cold PESK buffer SDS, sodium dodecyl sulfate. (40 mM potassium phosphate, 30 mM EDTA, 0.1 M 493 0003-9861/79/120493-07$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
494
NESS, SPINDLER,
sucrose, 50 mM potassium chloride, and 1 mM dithiothreitol at pH 7.2). The livers were minced with scissors to free the tissue of blood. The mince was then homogenized with 4 vol of cold PESK buffer using a motor-driven Potter-Elvehjem Teflon glass homogenizer. The resulting broken cell preparation was centrifuged at 10,500g for 15 min to remove mitochondria and cellular debris. The supernatant fraction was centrifuged for 60 min at 99,500g. The microsomal pellets were frozen and stored at -20°C for up to 10 days prior to solubilizing the enzyme. HMG-CoA reductase assay. Enzymic activity was determined by a radiochemical method. Reaction mixtures contained the following concentrations of components: 100 mM potassium phosphate buffer (pH 7.1); 2 mM dithiothreitol; 4 mM glucose 6-phosphate; 1 mM NADP+; 1 unit/ml of glucose g-phosphate dehydrogenase; 3.33 mg/ml of bovine serum albumin; 65 pM RS-[3-14C] HMG-CoA (96,000 dpm), and varying amounts of enzyme protein ranging from 0.002 to 120 pg. The final volume was 300 ~1. The enzyme was preincubated with all reaction mixture components except for HMG-CoA for 20 min at 37°C. The reactions were started by the addition of substrate. Incubation times ranged from 3 to 10 min. The reactions were terminated by the addition of 300 ~1 of 0.24~ HCl. To insure lactonization, the mixtures were incubated for an additional 20 min. The [‘“Cl mevalonolactone was separated from the unreacted substrate by anion-exchange chromatography using Pasteur pipet columns of Bio-Rad AG 1-x8 formate (10). In those cases where the enzyme preparations contained other enzymes which compete with the reductase for S-HMG-CoA (II), the [‘QZlmevalonolactone was isolated by thin-layer chromatography (12). The recovery of [L4C]mevalonate was calculated from the recovery of added [3H]mevalonate. The recovery averaged 68%. One unit of reductase activity is defined as that amount which converts 1 pmol of HMG-CoA to 1 pmol of mevalonate in 1 min under the conditions described above. Protein determinations. The protein concentration of microsomal suspensions was determined by a biuret method (13). Protein concentrations of all other enzyme fractions were routinely determined by the Coomassie dye binding method (14). This method was chosen because of its sensitivity and its lack of interference from compounds such as dithiothreitol and glycerol. For purposes of comparison, protein was also determined by the method of Lowry (15) after extensive dialysis of certain purified enzyme preparations. Bovine serum albumin was used as the standard for all methods. Polyacrglamide gel electrophoresis. Purified HMG CoA reductase was examined for purity in three different systems. Two gel systems were used to analyze the native enzyme. The first was that described by Williams and Reisfeld (16). In this system
AND MOFFLER 7.5% acrylamide gels pH 7.5 were used. The electrode buffer was Tris-barbital, pH 7.0. The second native gel system was that developed by Eichler (17). To prepare these gels three solutions were made. Solution A contained 28% (w/v) acrylamide and 0.56% (w/v) NJ’-methylene-bisacrylamide. Solution B contained 6.05% (w/v) Tris, 0.2% (v/v) N,N,N’,N’tetramethylethylenediamine (TEMED) and was adjusted to pH 8.9 with phosphoric acid. Solution C contained 0.14% (w/v) ammonium persulfate in 40% glycerol. One part of A, one part of B, and two parts of C were mixed and placed into gel tubes, overlayered with water, and allowed to polymerize at room temperature. The running buffer was 0.012~ glycine, pH 9.5. Electrophoresis was carried out at 4°C using 2 mA per gel. Following electrophoresis, the gels were either cut into slices for determination of enzyme activity or were stained with 0.1% Coomassie blue (R250) in methanol:water:acetic acid (10: lO:l, v/v/v) for 2 h at 37°C. Gels were destained with a solution of 7.5% acetic acid and 5% methanol. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed with 7.5% gels using a modification of the method of Weber and Osborne (18). The enzyme sample was dialyzed against 10 mM sodium phosphate buffer, pH 7.0. A 100~~1 aliquot of this preparation was mixed with 25 ~1 of 5% SDS, 0.2 M dithiothreitol in 0.205 M tris-acetate, pH 6.5. This mixture was heated in a boiling water bath for 5 min. Bromphenol blue was added and the samples were applied to the 7.5% gels which contained the same Tris-acetate buffer with 0.1% SDS. RESULTS
Solubilixation of HMG-CoA from Liver Microsomes
Reductase
The solubilization procedure is based on that described by Heller and Gould (18). Frozen microsomal pellets were allowed to thaw at room temperature. The pellets were homogenized in cold PESK buffer (12 ml per microsomal pellet derived from 7 g of liver). The suspension was centrifuged at 100,OOOg for 60 min at 4°C. The resulting supernatant solution was carefully removed with a Pasteur pipet. The pellet was frozen, thawed, and extracted four more times. A typical solubilization is shown in Table I. The third extract contained the largest amount of solubilized enzyme and had the highest specific activity. The amount of enzyme solubilized by this procedure ranged from 65 to 94% for 13 preparations with 5% or less remaining in the residual microsomes. Several investigators (4, 6, 7)
HYDROXYMETHYLGLLJTARYL
COENZYME TABLE
SOLUBILIZATION
A REDUCTASE
495
PURIFICATION
I
OF%HYDROXY-3-METHYLGLUTARYLCOENZYMEA
REDWCTASE
FROMRAT
LIVER
Extraction number
Activity (units)
Protein bg)
Specific activity
Percentage extracted
Microsomal suspension 1 2 3 4 5
8.62 1.55 1.87 2.57 1.11 0.30
2370 189 39 25 17 11
0.0037 0.0083 0.0475 0.1050 0.0665 0.0261
18.0 21.7 29.9 12.9 3.5
Total extract
7.40
281
0.0264
85.8
used one or two extractions and obtain from 30 to 67% solubilization of enzyme activity. Edwards et al. (8) recently described a method for solubilizing 190% of the activity originally present in the microsomes. The five extracts were combined and incubated at 37°C for 90 min. This incubation inactivates the enzymic activities which compete with the reductase for HMG-CoA (11). After the incubation the extract was centrifuged at 20,OOOg for 10 min to remove precipitated protein. The clear extract was then concentrated with ammonium sulfate and the 30 to 50% of saturation fraction
r
was dissolved in PESK buffer. This concentrated extract was then frozen at -20°C. Heat Treatment of Solubilized HMG-CoA Reductase
The concentrated extract was thawed at 37°C and glycerol and potassium chloride were added to give final concentrations of 33% and 1 M, respectively. Invariably the addition of glycerol and KC1 resulted in a slight activation, The enzyme was placed in prewarmed tubes and heated at 64°C for 10 min. After heating, the enzyme was I
I
I I - 0.25
FRACTION
NUMBER
1. Elution profile of HMG-CoA reductase from an Affi-Gel Blue column. Heat-treated enzyme, 4.76 units in a volume of 300 ml, was applied. Fractions of 7.5 ml were collected. At A the column was washed with 100 ml of PESK buffer containing 0.2 M KCl. At B a gradient of 0.2 to 2.0 M KC1 in PESK buffer was introduced. FIG.
496
NESS, SPINDLER,
rapidly cooled to room temperature using an ice-water bath. The enzyme was then centrifuged at 144,000g for 15 min to remove precipitated protein. This heat treatment resulted in a three- to fourfold purification with 85% or better recovery. AfJi-Gel Blue ChromatogralPhy
The heat-treated enzyme was diluted fivefold with PESK buffer to reduce the concentration of KC1 to 0.2 M. The diluted enzyme was applied to an Affi-Gel Blue column (1.5 x 4.0 cm) equilibrated with PESK buffer containing 0.2 M KC1 at room temperature. The column was washed with 100 ml of this buffer and then eluted with a linear gradient of KC1 from 0.2 to 2.0 M in PESK buffer. A typical elution profile is shown in Fig. 1. All fractions containing more than 0.05 units of activity were pooled and concentrated with an Amicon ultrafiltration cell using a YM-10 filter. The KC1 was removed by repeated ultrafiltration and addition of PESK buffer. Glycerol was then added to the enzyme in PESK to give a final glycerol concentration of 50% and the enzyme was stored at -70°C.
4
6
12 FRACTION
AND MOFFLER
HMG-CoA
Agarose Chromatography
The enzyme obtained after Affi-Gel Blue chromatography was thawed and diluted with water to give a glycerol concentration of 25%. The diluted enzyme solution was applied to an HMG-CoA agarose column (0.6 x 2.0 cm) equilibrated with PESK: glycerol:water (1:1:2) at room temperature. The column was then eluted sequentially with 40 ml of PESK:glycerol:water containing ‘70 IBM KCl; 10 ml of PESK:glycerol: water containing 70 mM KC1 and 200 PM RS-HMG-CoA and then PESK:glycerol: water containing 0.5 M KCl. As shown in Fig. 2, the majority of the reductase was eluted from the column by the addition of HMG-CoA to the elution buffer. A small amount of enzyme, usually less than 15%, was eluted with KC1 alone. Interestingly, a significant amount of protein was eluted from the column after the reductase. A typical purification of hepatic microsomal HMG-CoA reductase from 14 rats is summarized in Table II. The specific activities of eight different preparations ranged from 16.4 to 23.4 pmol of mevalonate formed/min/mg of protein. These specific
16
20
24
NUMBER
FIG. 2. Elution profile of HMGCoA reductase from a HMG-CoA agarose column. Enzyme from the Affi-Gel Blue step, 2.05 units in a volume of 38 ml, was applied. Four-milliliter fractions were collected. At A the column was washed with PESKglycerohwater (1:1:2) containing ‘70 mM KCI. At B 200 pM RS-HMGCoA was added to the eluting buffer. At C the KC1 concentration was increased to 0.5 M.
HYDROXYMETHYLGLUTARYL
COENZYME TABLE
A REDUCTASE
II
PURIFICATION OF%HYDROXY-3-METHYLGLUTARYLCOENZYME Purification step
Activity (units)
Microsomal suspension Concentrated extract Heat treatment, 64°C Affi-Gel Blue HMG-CoA agarose
Protein (mg)
497
PURIFICATION
Specific activity
A REDUCTASE FROM RAT LIVERY Purification (-fold)
Recovery (percent)
8.01
2900
0.003
1.0
6.11
185
0.033
11.6
76.3
0.129 1.61 19.2
46.2 576 6860
75.8 44.3 22.1
47 2.2 0.092
6.07 3.55 1.77
100
a Reductase was purified from 119 g of liver.
activities reported.
are higher
than any previously
Criteria of Purity
The enzyme which eluted from the HMGCoA agarose column in response to HMGCoA was concentrated and examined for purity by polyacrylamide gel electrophoresis. As shown in Fig. 3, the enzyme migrated
as a single band in both native and SDS polyacrylamide gel systems. When native gels were sliced and assayed for reductase activity, it was found that the enzymic activity coincided with the protein band (Fig. 4). Both gave Rfs of approximately 0.45 in this system. Protein and enzyme activity also comigrated using the neutral buffer system described by Williams and Reisfeld (16). Molecular
Weight Determination
The subunit molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 50,000 + 2,000 for five homogeneous enzyme preparations. This value is in good agreement with those reported by others (6-8). Since the molecular weight of the native enzyme as determined by several investigators (2, 4, 19) appears to be about 200,000, it is likely that the reductase is composed of four subunits. DISCUSSION
A
B
FIG. 3. Polyacrylamide gel electrophoresis of purified HMG-CoA reductase on native (A) and SDS gels (B). In A, 8 pg of enzyme was electrophoresed in the system developed by Eichler (17). In B, 14 pg of enzyme was electrophoresed in SDS as described under Experimental Procedures.
The procedure for the purification of HMG-CoA reductase described in this paper yields preparations with specific activities ranging from 16 to 23 pmol of mevalonate formed/min/mg of protein.2 This is the highest specific activity re* Recent studies in our laboratory indicate that purified HMG-CoA reductase can be activated nearly twofold either by incubation with microsomal lipids or by incubation with a phosphatase preparation from liver cytosol.
498 3 yc
NESS, SPINDLER, r
1
1
AND MOFFLER
zyme sample and elution buffers was required to maximize recovery of enzyme activity. P As pointed out in the introductory statez ment, several reports have appeared which 5 0.24claim to have developed purification procedures which yield homogeneous preparations of HMG-CoA reductase from rat liver (2-8). In all cases the specific activities are lower than those obtained in the present study. Many of these reports failed to show that reductase activity was associated with the single observable protein band on 0.2 0.4 0.6 0.0 polyacrylamide gels (4, 5, 7,8). Berde et al. Rf (9) demonstrated that reductase preparations obtained by the procedures of TorFIG. 4. Comigration of HMG-CoA reductase activmanen et al. (5) and Heller and Shrewsity and protein on polyacrylamide gels. Three micrograms of enzyme purified through the HMG-CoA bury (4) were actually not pure as reductase agarose step was applied to each of two identical 6% activity did not coincide with the major gels prepared according to the procedure of Eichler protein species on polyacrylamide gels in (17). Electrophoresis was performed at 5-W using either case. Srikantaiah et al. (6) have 2 mA per tube. After electrophoresis one gel was reported reductase activity associated with stained with Coomassie blue, destained, and photothe major protein band on polyacrylamide graphed while the other was sliced into 0.5cm gels. However, the specific activity of their sections and assayed for reductase activity. best preparation was 3.35 ~mol/minlmg which is less than 20% of that obtained in ported for the reductase. The enzyme prep- the present study. In addition, these inarations are essentially homogeneous as vestigators reported K,s for S-HMG-CoA judged by comigration of enzyme activity and NADPH of 34 and 320 PM, respectively. and protein on polyacrylamide gels (Fig. 4). The apparent K,s for S-HMG-CoA and The yield ranges from 10 to 25% with the NADPH of our homogeneous reductase single largest loss of activity occurring in preparations were 0.5 and 30 PM, respecthe HMG-CoA Agarose step. The enzyme tively. In a previous study (19) we dempreparations were stable for at least 3 onstrated that enzymes which compete with months when stored in 50% glycerol at the reductase for S-HMG-CoA are present -70°C. No significant loss of activity was in microsomal preparations and when these noted after freezing and thawing several competing enzymes are present, high K, times. values are observed. There are two steps in the purification There are several possible explanations procedure that are particularly critical. The for the higher specific activity of the enzyme first is the 90-min incubation of the extract. preparations described in this paper as comThis step eliminates other enzymes which pared with previous reports. One obvious compete with the reductase for S-HMG-CoA explanation is that previous preparations (11). The second occurs in the HMG-CoA were impure. A second explanation is that agarose step (Fig. 2) where it is essential previous preparations contained significant to completely remove the KC1 from the amounts of inactive enzyme. Kleinsek et al. sample and to dilute the PESK buffer to (7) reported that a protein fraction that bind the enzyme to the column. In addition did not bind to the coenzyme A affinity colit is necessary to use low concentrations umn was immunologically identical to the of KC1 (i.e., 50 to 80 mM) in order to selec- reductase. Also Colombo et al. (20) removed tively elute the reductase with HMG-CoA. an inactive form of phosphoenolpyruvate Finally, the inclusion of glyerol in the en- carboxykinase from their enzyme prepara0.2 < 0.32 - I ".$ .pas,
0.4
0.6
0.0
1.0 1
HYDROXYMETHYLGLUTARYL
COENZYME
tions by agarose-hexane-GTP affinity chromatography. An inactive form of the reductase could arise during purification due to oxidation of sulfhydryl groups or other modifications. Another possible explanation for differing specific activities for homogeneous preparations of HMG-CoA reductase could be differing amounts of bound lipids. Berde et al. (9) have shown a threefold activation of reductase activity by addition of phospholipids. Srikantaiah et al. (6) reported the purification of HMG-CoA reductase from normal and cholestyramine-fed rats. The enzyme from the cholestyraminefed animals had a three- to fourfold higher specific activity and contained 36% less cholesterol. They concluded that the decreased amount of cholesterol was responsible for the higher specific activity. Different specific activities for homogeneous reductase preparations can result from differences in methods used for assaying protein. Chiapelli et- al. (21) have shown that the Coomassie dye-binding method (14) consistently yields lower protein values than the Lowry method (15) when bovine serum albumin is used as the standard. This is due to the high color yield that bovine serum albumin gives in the dye-binding method. When the protein concentration obtained from the Lowry method was used in calculating the specific activity of a purified reductase preparation, this value decreased from 23.4 to 13.5 pmol/min/mg. The purification procedure described in this report should provide an important tool for studies of the mechanisms of regulation of HMG-CoA reductase activity and cholesterol biosynthesis.
ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Gary A. Benton, John P. Miller, and Wyndie J. Burriss. This work was supported by USPHS Grant HL 18094 awarded to Gene C. Ness.
A REDUCTASE
PURIFICATION
499
REFERENCES 1. DIETSCHY, J. M., AND BROWN, M. S. (1974) J. Lipid Res. 15, 508-516. 2. KAWACHI, T., AND RUDNEY, H. (1970) Biochemistry 9, 1700-1705. 3. HIGGINS, M. J. P., BRADY, D., AND RUDNEY, H. (1974) Arch. Biochem. Biophys. 163, 271-282. 4. HELLER, R. A., AND SHREWSBURY, M. A. (1976) J. Biol. Chem. 251, 3815-3822. 5. TORMANEN, C. D., REDD, W. L., SRIKANTAIAH, M. V., AND SCALLEN, T. J. (1976) Biochem. Biophys. Res. Commun. 68, 754-762. 6. SRIKANTAIAH, M. V., TORMANEN, C. D., REDD, W. L., HARDGRAVE, J. E., AND SCALLEN, T. J. (1977) J. Biol. Chem. 252, 6145-6150. 7. KLEINSEK, D. A., RANGANATHAN, S., AND PORTER, J. W. (1977) Proc. Nat. Acad. Sci. USA 74, 1431-1435. 8. Edwards, P. A., Lemongello, D., and Fogelman, A. M. (1979) J. Lipid Res. 20, 40-46. 9. BERDE, C. B., HELLER, R. A., AND SIMONI, R. D. (1977) Biochim. Biophys. Acta 488, 112-120. 10. AVIGAN, J., BHATHENA, S. J., AND SCHREINER, M. E. (1975) J. Lipid Res. 16, 151-154. 11. NESS, G. C., AND MOFFLER, M. H. (1978) Arch. Biochem. Biophys. 189, 221-223. 12. SHAPIRO, D. J., NORDSTROM, J. L., MITSCHELEN, J. J., RODWELL, V. W., AND SCHIMKE, R. T. (1975) Biochem. Biophys. Acta 370, 369-377. 13. LEE, Y. P., AND LARDY, H. A. (1965) J. Biol. Chem. 240, 1427- 1436. 14. BRADFORD, M. M. (1976)Anal. Biochem. 72,248254. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. WILLIAMS, D. E., AND REISFELD, R. A. (1964) Ann. N. Y. Acad. Sci. 121, 373-381. 17. EICHLER, D. C. (1973) Ph.D. Thesis, University of California at Los Angeles. 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 19. HELLER, R. A., AND GOULD, R. G. (19’75) Biochim. Biophys. Acta 388, 254-259. 20. Ness, G. C., and Moffler, M. H. (1979) Biochim. Biophys. Acta 572, 333-344. 21. COLOMBO, G., CARLSON, G. M., AND LARDY, H. A. (1978) Biochemistry 17, 5321-5329. 22. CHIAPPELLI, F., VASIL, A., AND HAGGERTY, D. F. (1979) Anal. Biochem. 94, 160-165.
AND BIOPHYSICS
pp. 493-499, 1979
Purification GENE
of 3-Hydroxy3-Methylglutaryl Reductase from Rat Liver
C. NESS, Department
CHRISTY
of Biochemistry,
D. SPINDLER, College Tampa,
Coenzyme
AND MARY
of Medicine, University Florida 33612
A
H. MOFFLER
of South
Florida,
Received February 22, 1979; revised June 7, 1979 A procedure for the purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.341 from rat liver microsomes has been developed. The enzyme preparations obtained by this procedure have specific activities of 16 to 23 pmol of mevalonate formed per minute per milligram of protein. These enzyme preparations were judged to be homogeneous on the basis of comigration of enzyme activity and protein on polyacrylamide gels.
The microsomal enzyme, 3-hydroxy-3methylglutaryl coenzyme A reductase, catalyzes the rate-limiting reaction in cholesterol biosynthesis (1). In order to gain a better understanding of the physiological mechanism(s) which regulate this enzyme, considerable effort has been devoted to purifying the reductase. Several reports have appeared (2-8) which describe procedures for obtaining homogeneous preparations of the reductase from rat liver. These procedures yield preparations with widely different specific activities and properties. Recently, it was shown that two of these previously reported homogeneous enzyme preparations (4, 5) were actually quite impure when analyzed by electrophoresis on polyacrylamide gels and assayed for enzyme activity as well as stained for protein (9). In this report we describe a purification procedure which sequentially utilizes Affi-Gel Blue and HMG-CoA agarose’
affinity chromatography. This procedure yields homogeneous enzyme preparations with specific activities of 16 to 23 pmol of mevalonate formed per minute per-milligram of protein. EXPERIMENTAL
PROCEDURE
Materials. RS-3-[3-14C-glutaryl]hydroxy-3-methylglutaryl coenzyme A was purchased from New England Nuclear. Agarose-hexane 3-hydroxy-3-methylglutaryl coenzyme A, Type 5 (HMG-CoA agarose) was purchased from P. L. Biochemicals. Affi-Gel Blue, 75-150 pm, Bio-Rad protein assay dye reagent concentrate, BioPhore 7.5% gels and AG l-x8 formate, 200-400 mesh were purchased from Bio-Rad Laboratories. The liquid scintillation cocktail, ACS, was purchased from Amersham. Cholestyramine (Questran) was purchased from Mead Johnson. Glucose 6-phosphate dehydrogenase (type XII), NADP+, NADPH, dithiothreitol, and unlabeled HMG-CoA were purchased from Sigma Chemical Company. Animals. Male Sprague-Dawley rats weighing 125-150 g were purchased from ARSiSpragueDawley, Madison, Wisconsin. The animals were housed in a light-controlled room in which the dark ’ Abbreviations used: HMG-CoA agarose, agaroseperiod was from 1000 to 2200. The animals were fed hexane 3-hydroxy-3-methylglutaryl coenzyme A; ground Wayne Lab Blox containing2% cholestyramine PESK buffer, 40 mM potassium phosphate, 30 mM for 6 to 8 days prior to being killed. Under these EDTA, 0.1 M sucrose, 50 mM potassium chloride, and conditions maximal HMG-CoA reductase activity oc1 mM dithiothreitol; solution A, 28% (w/v) a&amide curs at 3 h into the dark period which is referred to and 0.56% (w/v) N,N’-methylene-bisacrylamide; solu- as D-3. tion, B, 6.05% (w/v) Tris, 0.2% (v/v) N,N,N’,N’Preparation of microsomes. Cholestyramine-fed tetramethylethylenediamine (TEMED); solution C, rats were killed by decapitation at D-3 and their livers 0.14% (w/v) ammonium persulfate in 40% glycerol; were quickly removed and placed in cold PESK buffer SDS, sodium dodecyl sulfate. (40 mM potassium phosphate, 30 mM EDTA, 0.1 M 493 0003-9861/79/120493-07$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
494
NESS, SPINDLER,
sucrose, 50 mM potassium chloride, and 1 mM dithiothreitol at pH 7.2). The livers were minced with scissors to free the tissue of blood. The mince was then homogenized with 4 vol of cold PESK buffer using a motor-driven Potter-Elvehjem Teflon glass homogenizer. The resulting broken cell preparation was centrifuged at 10,500g for 15 min to remove mitochondria and cellular debris. The supernatant fraction was centrifuged for 60 min at 99,500g. The microsomal pellets were frozen and stored at -20°C for up to 10 days prior to solubilizing the enzyme. HMG-CoA reductase assay. Enzymic activity was determined by a radiochemical method. Reaction mixtures contained the following concentrations of components: 100 mM potassium phosphate buffer (pH 7.1); 2 mM dithiothreitol; 4 mM glucose 6-phosphate; 1 mM NADP+; 1 unit/ml of glucose g-phosphate dehydrogenase; 3.33 mg/ml of bovine serum albumin; 65 pM RS-[3-14C] HMG-CoA (96,000 dpm), and varying amounts of enzyme protein ranging from 0.002 to 120 pg. The final volume was 300 ~1. The enzyme was preincubated with all reaction mixture components except for HMG-CoA for 20 min at 37°C. The reactions were started by the addition of substrate. Incubation times ranged from 3 to 10 min. The reactions were terminated by the addition of 300 ~1 of 0.24~ HCl. To insure lactonization, the mixtures were incubated for an additional 20 min. The [‘“Cl mevalonolactone was separated from the unreacted substrate by anion-exchange chromatography using Pasteur pipet columns of Bio-Rad AG 1-x8 formate (10). In those cases where the enzyme preparations contained other enzymes which compete with the reductase for S-HMG-CoA (II), the [‘QZlmevalonolactone was isolated by thin-layer chromatography (12). The recovery of [L4C]mevalonate was calculated from the recovery of added [3H]mevalonate. The recovery averaged 68%. One unit of reductase activity is defined as that amount which converts 1 pmol of HMG-CoA to 1 pmol of mevalonate in 1 min under the conditions described above. Protein determinations. The protein concentration of microsomal suspensions was determined by a biuret method (13). Protein concentrations of all other enzyme fractions were routinely determined by the Coomassie dye binding method (14). This method was chosen because of its sensitivity and its lack of interference from compounds such as dithiothreitol and glycerol. For purposes of comparison, protein was also determined by the method of Lowry (15) after extensive dialysis of certain purified enzyme preparations. Bovine serum albumin was used as the standard for all methods. Polyacrglamide gel electrophoresis. Purified HMG CoA reductase was examined for purity in three different systems. Two gel systems were used to analyze the native enzyme. The first was that described by Williams and Reisfeld (16). In this system
AND MOFFLER 7.5% acrylamide gels pH 7.5 were used. The electrode buffer was Tris-barbital, pH 7.0. The second native gel system was that developed by Eichler (17). To prepare these gels three solutions were made. Solution A contained 28% (w/v) acrylamide and 0.56% (w/v) NJ’-methylene-bisacrylamide. Solution B contained 6.05% (w/v) Tris, 0.2% (v/v) N,N,N’,N’tetramethylethylenediamine (TEMED) and was adjusted to pH 8.9 with phosphoric acid. Solution C contained 0.14% (w/v) ammonium persulfate in 40% glycerol. One part of A, one part of B, and two parts of C were mixed and placed into gel tubes, overlayered with water, and allowed to polymerize at room temperature. The running buffer was 0.012~ glycine, pH 9.5. Electrophoresis was carried out at 4°C using 2 mA per gel. Following electrophoresis, the gels were either cut into slices for determination of enzyme activity or were stained with 0.1% Coomassie blue (R250) in methanol:water:acetic acid (10: lO:l, v/v/v) for 2 h at 37°C. Gels were destained with a solution of 7.5% acetic acid and 5% methanol. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed with 7.5% gels using a modification of the method of Weber and Osborne (18). The enzyme sample was dialyzed against 10 mM sodium phosphate buffer, pH 7.0. A 100~~1 aliquot of this preparation was mixed with 25 ~1 of 5% SDS, 0.2 M dithiothreitol in 0.205 M tris-acetate, pH 6.5. This mixture was heated in a boiling water bath for 5 min. Bromphenol blue was added and the samples were applied to the 7.5% gels which contained the same Tris-acetate buffer with 0.1% SDS. RESULTS
Solubilixation of HMG-CoA from Liver Microsomes
Reductase
The solubilization procedure is based on that described by Heller and Gould (18). Frozen microsomal pellets were allowed to thaw at room temperature. The pellets were homogenized in cold PESK buffer (12 ml per microsomal pellet derived from 7 g of liver). The suspension was centrifuged at 100,OOOg for 60 min at 4°C. The resulting supernatant solution was carefully removed with a Pasteur pipet. The pellet was frozen, thawed, and extracted four more times. A typical solubilization is shown in Table I. The third extract contained the largest amount of solubilized enzyme and had the highest specific activity. The amount of enzyme solubilized by this procedure ranged from 65 to 94% for 13 preparations with 5% or less remaining in the residual microsomes. Several investigators (4, 6, 7)
HYDROXYMETHYLGLLJTARYL
COENZYME TABLE
SOLUBILIZATION
A REDUCTASE
495
PURIFICATION
I
OF%HYDROXY-3-METHYLGLUTARYLCOENZYMEA
REDWCTASE
FROMRAT
LIVER
Extraction number
Activity (units)
Protein bg)
Specific activity
Percentage extracted
Microsomal suspension 1 2 3 4 5
8.62 1.55 1.87 2.57 1.11 0.30
2370 189 39 25 17 11
0.0037 0.0083 0.0475 0.1050 0.0665 0.0261
18.0 21.7 29.9 12.9 3.5
Total extract
7.40
281
0.0264
85.8
used one or two extractions and obtain from 30 to 67% solubilization of enzyme activity. Edwards et al. (8) recently described a method for solubilizing 190% of the activity originally present in the microsomes. The five extracts were combined and incubated at 37°C for 90 min. This incubation inactivates the enzymic activities which compete with the reductase for HMG-CoA (11). After the incubation the extract was centrifuged at 20,OOOg for 10 min to remove precipitated protein. The clear extract was then concentrated with ammonium sulfate and the 30 to 50% of saturation fraction
r
was dissolved in PESK buffer. This concentrated extract was then frozen at -20°C. Heat Treatment of Solubilized HMG-CoA Reductase
The concentrated extract was thawed at 37°C and glycerol and potassium chloride were added to give final concentrations of 33% and 1 M, respectively. Invariably the addition of glycerol and KC1 resulted in a slight activation, The enzyme was placed in prewarmed tubes and heated at 64°C for 10 min. After heating, the enzyme was I
I
I I - 0.25
FRACTION
NUMBER
1. Elution profile of HMG-CoA reductase from an Affi-Gel Blue column. Heat-treated enzyme, 4.76 units in a volume of 300 ml, was applied. Fractions of 7.5 ml were collected. At A the column was washed with 100 ml of PESK buffer containing 0.2 M KCl. At B a gradient of 0.2 to 2.0 M KC1 in PESK buffer was introduced. FIG.
496
NESS, SPINDLER,
rapidly cooled to room temperature using an ice-water bath. The enzyme was then centrifuged at 144,000g for 15 min to remove precipitated protein. This heat treatment resulted in a three- to fourfold purification with 85% or better recovery. AfJi-Gel Blue ChromatogralPhy
The heat-treated enzyme was diluted fivefold with PESK buffer to reduce the concentration of KC1 to 0.2 M. The diluted enzyme was applied to an Affi-Gel Blue column (1.5 x 4.0 cm) equilibrated with PESK buffer containing 0.2 M KC1 at room temperature. The column was washed with 100 ml of this buffer and then eluted with a linear gradient of KC1 from 0.2 to 2.0 M in PESK buffer. A typical elution profile is shown in Fig. 1. All fractions containing more than 0.05 units of activity were pooled and concentrated with an Amicon ultrafiltration cell using a YM-10 filter. The KC1 was removed by repeated ultrafiltration and addition of PESK buffer. Glycerol was then added to the enzyme in PESK to give a final glycerol concentration of 50% and the enzyme was stored at -70°C.
4
6
12 FRACTION
AND MOFFLER
HMG-CoA
Agarose Chromatography
The enzyme obtained after Affi-Gel Blue chromatography was thawed and diluted with water to give a glycerol concentration of 25%. The diluted enzyme solution was applied to an HMG-CoA agarose column (0.6 x 2.0 cm) equilibrated with PESK: glycerol:water (1:1:2) at room temperature. The column was then eluted sequentially with 40 ml of PESK:glycerol:water containing ‘70 IBM KCl; 10 ml of PESK:glycerol: water containing 70 mM KC1 and 200 PM RS-HMG-CoA and then PESK:glycerol: water containing 0.5 M KCl. As shown in Fig. 2, the majority of the reductase was eluted from the column by the addition of HMG-CoA to the elution buffer. A small amount of enzyme, usually less than 15%, was eluted with KC1 alone. Interestingly, a significant amount of protein was eluted from the column after the reductase. A typical purification of hepatic microsomal HMG-CoA reductase from 14 rats is summarized in Table II. The specific activities of eight different preparations ranged from 16.4 to 23.4 pmol of mevalonate formed/min/mg of protein. These specific
16
20
24
NUMBER
FIG. 2. Elution profile of HMGCoA reductase from a HMG-CoA agarose column. Enzyme from the Affi-Gel Blue step, 2.05 units in a volume of 38 ml, was applied. Four-milliliter fractions were collected. At A the column was washed with PESKglycerohwater (1:1:2) containing ‘70 mM KCI. At B 200 pM RS-HMGCoA was added to the eluting buffer. At C the KC1 concentration was increased to 0.5 M.
HYDROXYMETHYLGLUTARYL
COENZYME TABLE
A REDUCTASE
II
PURIFICATION OF%HYDROXY-3-METHYLGLUTARYLCOENZYME Purification step
Activity (units)
Microsomal suspension Concentrated extract Heat treatment, 64°C Affi-Gel Blue HMG-CoA agarose
Protein (mg)
497
PURIFICATION
Specific activity
A REDUCTASE FROM RAT LIVERY Purification (-fold)
Recovery (percent)
8.01
2900
0.003
1.0
6.11
185
0.033
11.6
76.3
0.129 1.61 19.2
46.2 576 6860
75.8 44.3 22.1
47 2.2 0.092
6.07 3.55 1.77
100
a Reductase was purified from 119 g of liver.
activities reported.
are higher
than any previously
Criteria of Purity
The enzyme which eluted from the HMGCoA agarose column in response to HMGCoA was concentrated and examined for purity by polyacrylamide gel electrophoresis. As shown in Fig. 3, the enzyme migrated
as a single band in both native and SDS polyacrylamide gel systems. When native gels were sliced and assayed for reductase activity, it was found that the enzymic activity coincided with the protein band (Fig. 4). Both gave Rfs of approximately 0.45 in this system. Protein and enzyme activity also comigrated using the neutral buffer system described by Williams and Reisfeld (16). Molecular
Weight Determination
The subunit molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 50,000 + 2,000 for five homogeneous enzyme preparations. This value is in good agreement with those reported by others (6-8). Since the molecular weight of the native enzyme as determined by several investigators (2, 4, 19) appears to be about 200,000, it is likely that the reductase is composed of four subunits. DISCUSSION
A
B
FIG. 3. Polyacrylamide gel electrophoresis of purified HMG-CoA reductase on native (A) and SDS gels (B). In A, 8 pg of enzyme was electrophoresed in the system developed by Eichler (17). In B, 14 pg of enzyme was electrophoresed in SDS as described under Experimental Procedures.
The procedure for the purification of HMG-CoA reductase described in this paper yields preparations with specific activities ranging from 16 to 23 pmol of mevalonate formed/min/mg of protein.2 This is the highest specific activity re* Recent studies in our laboratory indicate that purified HMG-CoA reductase can be activated nearly twofold either by incubation with microsomal lipids or by incubation with a phosphatase preparation from liver cytosol.
498 3 yc
NESS, SPINDLER, r
1
1
AND MOFFLER
zyme sample and elution buffers was required to maximize recovery of enzyme activity. P As pointed out in the introductory statez ment, several reports have appeared which 5 0.24claim to have developed purification procedures which yield homogeneous preparations of HMG-CoA reductase from rat liver (2-8). In all cases the specific activities are lower than those obtained in the present study. Many of these reports failed to show that reductase activity was associated with the single observable protein band on 0.2 0.4 0.6 0.0 polyacrylamide gels (4, 5, 7,8). Berde et al. Rf (9) demonstrated that reductase preparations obtained by the procedures of TorFIG. 4. Comigration of HMG-CoA reductase activmanen et al. (5) and Heller and Shrewsity and protein on polyacrylamide gels. Three micrograms of enzyme purified through the HMG-CoA bury (4) were actually not pure as reductase agarose step was applied to each of two identical 6% activity did not coincide with the major gels prepared according to the procedure of Eichler protein species on polyacrylamide gels in (17). Electrophoresis was performed at 5-W using either case. Srikantaiah et al. (6) have 2 mA per tube. After electrophoresis one gel was reported reductase activity associated with stained with Coomassie blue, destained, and photothe major protein band on polyacrylamide graphed while the other was sliced into 0.5cm gels. However, the specific activity of their sections and assayed for reductase activity. best preparation was 3.35 ~mol/minlmg which is less than 20% of that obtained in ported for the reductase. The enzyme prep- the present study. In addition, these inarations are essentially homogeneous as vestigators reported K,s for S-HMG-CoA judged by comigration of enzyme activity and NADPH of 34 and 320 PM, respectively. and protein on polyacrylamide gels (Fig. 4). The apparent K,s for S-HMG-CoA and The yield ranges from 10 to 25% with the NADPH of our homogeneous reductase single largest loss of activity occurring in preparations were 0.5 and 30 PM, respecthe HMG-CoA Agarose step. The enzyme tively. In a previous study (19) we dempreparations were stable for at least 3 onstrated that enzymes which compete with months when stored in 50% glycerol at the reductase for S-HMG-CoA are present -70°C. No significant loss of activity was in microsomal preparations and when these noted after freezing and thawing several competing enzymes are present, high K, times. values are observed. There are two steps in the purification There are several possible explanations procedure that are particularly critical. The for the higher specific activity of the enzyme first is the 90-min incubation of the extract. preparations described in this paper as comThis step eliminates other enzymes which pared with previous reports. One obvious compete with the reductase for S-HMG-CoA explanation is that previous preparations (11). The second occurs in the HMG-CoA were impure. A second explanation is that agarose step (Fig. 2) where it is essential previous preparations contained significant to completely remove the KC1 from the amounts of inactive enzyme. Kleinsek et al. sample and to dilute the PESK buffer to (7) reported that a protein fraction that bind the enzyme to the column. In addition did not bind to the coenzyme A affinity colit is necessary to use low concentrations umn was immunologically identical to the of KC1 (i.e., 50 to 80 mM) in order to selec- reductase. Also Colombo et al. (20) removed tively elute the reductase with HMG-CoA. an inactive form of phosphoenolpyruvate Finally, the inclusion of glyerol in the en- carboxykinase from their enzyme prepara0.2 < 0.32 - I ".$ .pas,
0.4
0.6
0.0
1.0 1
HYDROXYMETHYLGLUTARYL
COENZYME
tions by agarose-hexane-GTP affinity chromatography. An inactive form of the reductase could arise during purification due to oxidation of sulfhydryl groups or other modifications. Another possible explanation for differing specific activities for homogeneous preparations of HMG-CoA reductase could be differing amounts of bound lipids. Berde et al. (9) have shown a threefold activation of reductase activity by addition of phospholipids. Srikantaiah et al. (6) reported the purification of HMG-CoA reductase from normal and cholestyramine-fed rats. The enzyme from the cholestyraminefed animals had a three- to fourfold higher specific activity and contained 36% less cholesterol. They concluded that the decreased amount of cholesterol was responsible for the higher specific activity. Different specific activities for homogeneous reductase preparations can result from differences in methods used for assaying protein. Chiapelli et- al. (21) have shown that the Coomassie dye-binding method (14) consistently yields lower protein values than the Lowry method (15) when bovine serum albumin is used as the standard. This is due to the high color yield that bovine serum albumin gives in the dye-binding method. When the protein concentration obtained from the Lowry method was used in calculating the specific activity of a purified reductase preparation, this value decreased from 23.4 to 13.5 pmol/min/mg. The purification procedure described in this report should provide an important tool for studies of the mechanisms of regulation of HMG-CoA reductase activity and cholesterol biosynthesis.
ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Gary A. Benton, John P. Miller, and Wyndie J. Burriss. This work was supported by USPHS Grant HL 18094 awarded to Gene C. Ness.
A REDUCTASE
PURIFICATION
499
REFERENCES 1. DIETSCHY, J. M., AND BROWN, M. S. (1974) J. Lipid Res. 15, 508-516. 2. KAWACHI, T., AND RUDNEY, H. (1970) Biochemistry 9, 1700-1705. 3. HIGGINS, M. J. P., BRADY, D., AND RUDNEY, H. (1974) Arch. Biochem. Biophys. 163, 271-282. 4. HELLER, R. A., AND SHREWSBURY, M. A. (1976) J. Biol. Chem. 251, 3815-3822. 5. TORMANEN, C. D., REDD, W. L., SRIKANTAIAH, M. V., AND SCALLEN, T. J. (1976) Biochem. Biophys. Res. Commun. 68, 754-762. 6. SRIKANTAIAH, M. V., TORMANEN, C. D., REDD, W. L., HARDGRAVE, J. E., AND SCALLEN, T. J. (1977) J. Biol. Chem. 252, 6145-6150. 7. KLEINSEK, D. A., RANGANATHAN, S., AND PORTER, J. W. (1977) Proc. Nat. Acad. Sci. USA 74, 1431-1435. 8. Edwards, P. A., Lemongello, D., and Fogelman, A. M. (1979) J. Lipid Res. 20, 40-46. 9. BERDE, C. B., HELLER, R. A., AND SIMONI, R. D. (1977) Biochim. Biophys. Acta 488, 112-120. 10. AVIGAN, J., BHATHENA, S. J., AND SCHREINER, M. E. (1975) J. Lipid Res. 16, 151-154. 11. NESS, G. C., AND MOFFLER, M. H. (1978) Arch. Biochem. Biophys. 189, 221-223. 12. SHAPIRO, D. J., NORDSTROM, J. L., MITSCHELEN, J. J., RODWELL, V. W., AND SCHIMKE, R. T. (1975) Biochem. Biophys. Acta 370, 369-377. 13. LEE, Y. P., AND LARDY, H. A. (1965) J. Biol. Chem. 240, 1427- 1436. 14. BRADFORD, M. M. (1976)Anal. Biochem. 72,248254. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. WILLIAMS, D. E., AND REISFELD, R. A. (1964) Ann. N. Y. Acad. Sci. 121, 373-381. 17. EICHLER, D. C. (1973) Ph.D. Thesis, University of California at Los Angeles. 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 19. HELLER, R. A., AND GOULD, R. G. (19’75) Biochim. Biophys. Acta 388, 254-259. 20. Ness, G. C., and Moffler, M. H. (1979) Biochim. Biophys. Acta 572, 333-344. 21. COLOMBO, G., CARLSON, G. M., AND LARDY, H. A. (1978) Biochemistry 17, 5321-5329. 22. CHIAPPELLI, F., VASIL, A., AND HAGGERTY, D. F. (1979) Anal. Biochem. 94, 160-165.
