[55] 3-Hydroxy-3-methylglutaryl-CoA reductase from rat liver

462

HYDROXY METHYLGLUTARYL-CoA ENZYMES

[55] 3 - H y d r o x y - 3 - M e t h y l g l u t a r y l - C o A Liver

[55]

Reductase from Rat

B y DON A . KLEINSEK, RICHARD E . D U G A N , TERRY A . BAKER, a n d JOHN W. PORTER CHs I

HOOC--CH2--C--CHz--CO--SCoA

+ 2NADPH

+ 2H +

!

OH

CHs I

HOOC--CH2--1C--CH2--CHzOH

+ 2NADP +

+ CoASH

OH

The two-step reduction of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) to mevalonate, the rate-determining step in hepatic cholesterol biosynthesis, is catalyzed by microsomal HMG-CoA reductase '-3 [EC 1.1.1.34 mevalonate : N A D P ÷ oxidoreductase (CoA-acylating)]. As expected from the central role of this enzyme in cholesterogenesis, its activity varies markedly as a function of the nutritional or hormonal state of the animal. 4-1a As a first step to a better understanding of the molecular mechanism(s) of control of this activity, it is necessary to obtain homogeneous HMG-CoA reductase. In this chapter we describe purification procedures for the enzyme that result in homogeneous or nearly homogeneous preparations of high specific activity. A number of purification procedures have been reported for rat liver i M. D. Siperstein and V. M. Fagan, J. Biol. Chem. 241, 602 (1966). 2 L. W. White and H. Rudney, Biochemistry 9, 2725 (1970). z D. Shapiro and V. W. Rodwell, Biochemistry 11, 1042 (1972). 4 M. E. Dempsey, Annu. Rev. Biochem. 43, 967 (1974). 5 M. D. Siperstein, Curr. Top. Cell. Regul. 2, 65 (1970). 6 V. W. Rodwell, J. L. Nordstrom, and J. J. Mitschelefi, Adv. Lipid Res. 14, 2 (1976). r A. A. Kandutsch, H. W. Chen, and H. J. Heiniger, Science 201, 498 (1978). 8 R. E. Dugan and J. W. Porter, in "The B i o c h e m i c a l A c t i o n s o f H o r m o n e s " (G. Litwack, ed.), Vol. 4, p. 197. A c a d e m i c Press, New York, 1977. 9 j. M. Dietschy and J. D. Wilson, N. Engl. J. Med. 282, 1128, 1179, 1241 (1970). 10 V. W. Rodwell, D. J. McNamara, and D. J. Shapiro, Adv. Enzymol. 38, 373 (1973). 11 W. M. Bortz, Metab. Clin. Exp. 22, 1507 (1973). 12 M. S. Brown and J. L. Goldstein, Science 191, 150 (1976). ~a j. L. Goldstein and M. S. Brown, Annu. Rev. Biochem. 46, 897 (1977).

METHODSIN ENZYMOLOGY.VOL. 71

Copyright © 1981by Academic Press, Inc. All rights of reproductionin any form reserved.

ISBN 0-12-181971-X

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463

H M G - C o A reductase. 14-z~ H o w e v e r , these procedures do not yield homogeneous preparations. 2z Recently, homogeneous enzyme o f high specific activity has been obtained by combining conventional protein purification steps with affinity chromatography. A description o f these techniques, presented below, is divided into two sections. The first section describes two purification procedures used to produce homogeneous e n z y m e of high specific activity that employ standard purification steps and an affinity chromatographic separation. A description of the assay procedures and the properties o f the e n z y m e is included in this section. The second section describes three purification procedures which produce enzyme o f high specific activity by employing similar standard purification steps plus two successive affinity chromatographic fractionation steps. S e l e c t e d Materials a n d R e a g e n t s

Chemicals and reagents were obtained from the following sources: 3-hydroxy-3-methylglutaric acid (Methods 1 and 2), Schwarz/Mann and (Method 4) Sigma Chemical Co.; 3-hydroxy-3-methyl [3-a4C]glutaryl-CoA (Method 4), 3-hydroxy-3-methyl [3-14C]glutaric acid (Methods 1-3, and 5), N e w England Nuclear; BioGel A-0.5m, 200-400 mesh (Method 1), Affi-Gel Blue (Methods 4 and 5), Bio-Rad protein dye concentrate (Method 4), Bio-Rad Laboratories; cholestyramine (Questran), Mead Johnson Laboratories; blue Sepharose CL-6B (Method 3), Pharmacia; agaroseh e x a n e - C o A , type 5 (Methods 1-3), agarose-hexane-HMG-CoA, type 5 (Methods 4 and 5), P-L Biochemicals, Inc. Assay Procedures and Properties; Purification by Methods 1 and 2 Procedure of Assay for E n z y m e Activity M e t h o d s l, 2, a n d 5 R a d i o c h e m i c a l M e t h o d o f A s s a y . The activities of microsomal and sol-

ubilized H M G - C o A reductase are determined by measurement of the ~4T. Kawachi and H. Rudney, Biochemistry 9, 1700 (1970). ~ M. Higgins, T. Kawachi, and H. Rudney, Biochem. Biophys. Res. Commun. 45, 138 (1971). 10M. J. P. Higgins, D. Brady, and H. Rudney, Arch. Biochem. Biophys. 163, 271 (1974). 17M. S. Brown, S. E. Dana, J. M. Dietschy, and M. D. Siperstein, J. Biol. Chem. 248, 4731 ( 1973). ~8R. A. Heller and R. G. Gould, Biochem. Biophys. Res. Commun. 50, 859 (1973). ~9R. A. Heller and M. A. Shrewsbury, J. Biol. Chem. 251, 3815 (1976). 20C. D. Tormanen, W. L. Redd, M. V. Srikantaiah, and T. J. Scallen, Biochem. Biophys. Res. Commun. 68, 754 (1976). 2~M. V. Srikantaiah, C. D. Tormanen, W. L. Redd, J. E. Hardgrave, and T. J. Scallen, J. Biol. Chem. 252, 6145 (1977). 22C. B. Berde, R. A. Heller, and R. D. Simoni, Biochim. Biophys. Acta 488, 112 (1977).

464

H Y D R O X Y M E T H Y L G L U T A R Y L - C o A ENZYMES

[55]

conversion of [3-14C]HMG-CoA to mevalonate in an assay mixture that employs a NADPH-generating system. 23 The reaction mixture in this assay contains potassium phosphate buffer, pH 7.0, 50 /zmol; dithiothreitol, 2/zmol; glucose 6-phosphate, 2/zmol; NADP ÷, 0.5/~mol; OL-[314C]HMG-CoA, 0.15 /.tmol; glucose-6-phosphate dehydrogenase, 1.25 units; and protein, 300--1200/~g, in a total volume of 0.5 ml. This reaction mixture is preincubated at 37° for 10 min with all components except HMG-CoA reductase. The reaction is then carded out for 10 min at 37° following the addition of microsomes. The assay for enzyme activity is linear at protein concentrations of 600-2400 /~g/ml; however, the measurement of the activity of solubilized enzyme is linear with lesser amounts. The reaction is terminated by the addition of 50/~1 of 2.4 M HCI, and the sample is incubated at 37° for 30 min to allow for the formation of mevalonolactone. To determine background radioactivity, acid is added to control tubes prior to the addition of enzyme. The reaction mixture is then centrifuged (tabletop centrifuge) to remove denatured protein. An aliquot of 200/zl of the reaction mixture is spotted on activated (110°, 30 min) silica gel G thin-layer plates and the chromatogram is developed in benzene-acetone (1 : I, v/v). The chromatogram is then divided into 2 × 2 cm sections, and the appropriate sections scraped into vials for assay of radioactivity. Spectrophotometric Method of Assay. 24 The HMG-CoA reductase activity in solubilized fractions is assayed spectrophotometrically by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADPH. The reaction mixture in a volume of 0.5 ml contains potassium phosphate buffer, pH 7.0, 50 ~mol; dithiothreitol, 2 /zmol; NADPH, 0.3 ~mol; DL-HMG-CoA, 0.15/.tmol; and enzyme, 0.2-400/xg of protein. The reaction mixture is preincubated in a 2-mm light path glass cuvette without HMG-CoA present for 5 min at 37°. The assay for enzyme activity is then carried out by the addition of HMG-CoA to the reaction mixture at 37° in a recording spectrophotometer. The initial velocity of the reaction is measured, and the net rate of NADPH oxidation is determined by subtracting the rate of its oxidation in the absence of HMG-CoA from the rate observed with both substrates present. Microsomal activity cannot be assayed by this procedure owing to the low HMG-CoA reductase activity and the turbidity produced by the particulate membrane fraction in the incubation medium. However, the 2a C. M. Nepokroeff, M. R. Lakshmanan, G. C. Ness, R. E. Dugan, and J. W. Porter, Arch. Biochem. Biophys. 160, 387 (1974). 24 D. A. Kleinsek, S. Ranganathan, and J. W. Porter, Proc. Natl. Acad. Sci. U.S.A. 74, 1431 (1977).

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465

spectral method is a more rapid, convenient, and economical (though less sensitive) means of measuring soluble enzyme activity than the radiochemical procedure. Units. One unit of enzyme activity is that quantity of enzyme that oxidizes 1 nmol of NADPH per minute at 37° under the above assay conditions. Specific activity is expressed in activity units per milligram of protein. Protein Determination. All protein solutions are treated with 10% (w/v) trichloroacetic acid. The precipitate obtained with microsomes is assayed for protein by a modification of the biuret method 25 that employs deoxycholate (40 mM). Solubilized enzyme preparations are assayed for protein content by the method of Lowry et al. 26

Preparation of Enzyme Method 124 Treatment of Animals. Male albino Holtzman rats are acclimated to an alternate 12-hr light-dark cycle for a period of 2-3 weeks. The animals, weighing 180-200 g, are fed ad libitum a 2% cholestyramine Wayne Lab Blox powdered diet for a minimum of 4 days prior to sacrifice at the mid-dark period, which is the diurnal high point of enzyme activity. Preparation of Microsomes. After the animals are decapitated livers are excised and immediately placed in an ice-cold homogenization buffer. All solutions used in the purification scheme contain deionized water, and all further operations for the isolation of the microsomes are carded out at 4°. The homogenization medium contains 50 mM potassium phosphate buffer, pH 7.0, 0.2 M sucrose, and 2 mM dithiothreitol (buffer A). The livers are homogenized in buffer A (2 ml per gram of liver) in a Waring blender at full speed for 15 sec, followed by three strokes with a motor-driven Teflon pestle in a Potter-Elvehjem type glass homogenizer. The homogenate is centrifuged at 15,000 g for 10 min, and the postmitochondrial supernatant solution is retained and centrifuged at 100,000 g for 75 min in 30-ml ultracentrifuge tubes filled to near capacity. The supernatant solution is decanted, and the white lipid-like material remaining on the ultracentrifugation tube wall is removed with cotton-tipped applicators. The microsomes are washed by resuspension of the pellet in buffer A containing 50 mM EDTA (1 ml per gram of liver) and homogenized as before. The 25 A. G. Gornali, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). 28 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

466

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[5S]

homogenate is centrifuged at 100,000 g for 60 min in tubes of the same size, and the washed microsomal pellets are slow-frozen by placing the ultracentrifuge tubes in a - 2 0 ° freezer. Each tube should contain the material obtained from approximately 1.5 rat livers. Solubilization of Enzyme. Microsomal pellets are kept frozen at - 2 0 ° for a minimum of 2 hr or may be stored at this temperature for several weeks without loss of enzyme activity. The microsomal pellets are thawed at room temperature, and a slight modification of the method of Heller and Gould 18 is used to liberate the enzyme from the membrane matrix. Three milliliters of buffer B (50 mM potassium phosphate, pH 7.0, 0.1 M sucrose, 2 mM dithiothreitol, 50 mM KCI, and 30 mM EDTA) are added to each microsomal pellet and homogenization is effected with three strokes of a tight-fitting Teflon pestle in a Potter-Elvehjem type glass homogenizer. This procedure is followed by an additional three strokes of the microsomal suspension, after dilution with 7 ml of buffer. Excessive heat produced during the homogenization is avoided by surrounding the glass homogenizer with cold water. After standing for 15-30 min at room temperature, the suspension is centrifuged at 100,000 g for 60 min at 20°. The supernatant solution, containing soluble HMG-CoA reductase, is saved and used for purification of the enzyme. All further operations are carried out at room temperature unless otherwise indicated. Salt Fractionation. A filtered saturated solution of ammonium sulfate is added dropwise at room temperature under a stream of nitrogen to a slow-stirring solution of soluble extract. The protein fraction precipitating between 35 and 50% saturation with ammonium sulfate is collected by low-speed centrifugation. Heat Treatment. The protein pellet obtained above is dissolved in heat-treatment buffer [50 mM potassium phosphate, pH 7.0, 3 mM dithiothreitol, 1.0 M KC1, and 30% (v/v) glycerol]. The concentration of protein before the heat treatment should be between 4 and 10 mg/ml, since protein concentrations higher than this result in a viscous solution that is difficult to centrifuge. The enzyme solution, 4-5 ml, is placed in a Pyrex glass tube (16 × 125 mm). 'After heating at 65° for 6 min in a water bath, the enzyme solution is rapidly cooled on ice to room temperature and then centrifuged at 100,000 g for 30 min to remove denatured protein. Enzyme Concentration. The supernatant solution is diluted 1:1 with buffer B and subjected to a fractionation by 0 to 50% saturation with ammonium sulfate. After centrifugation at 10,000 g for 5 min, the protein pellet is dissolved in a minimal volume of either buffer C or D, depending on whether a BioGel filtration or a sucrose density gradient ultracentrifugation step is used. BioGel Filtration. The protein pellet is dissolved in a volume of 1-2 ml

[55]

RAT LIVER H M G - C o A

REDUCTASE

467

of buffer C (50 mM potassium phosphate, pH 7.0, 2 mM dithiothreitol, 30 mM EDTA, 50 mM KC1, and 10% sucrose) and then 4000 units of enzyme activity are applied to a BioGel A-0.5m gel filtration column (2 x 44 cm) having a particle size of 200-400 mesh. The enzyme is eluted at a flow rate of 6--9 ml/hr with the same buffer. About 80 eluate fractions (each 1 ml) are collected and analyzed for protein and enzyme activity. Those fractions that have minimal light absorbance at 280 nm and contain high enzyme activity are pooled for affinity chromatography. The gel may be used several times by washing the column between preparations with a buffer containing 50 mM potassium phosphate, pH 7.0, and 0.5 M KC1. The success of BioGel filtration is dependent upon the batch of agarose gel and the ionic strength of the eluent buffer. 27 Use of a high ionic strength eluent buffer, or a g d in which an interaction with the enzyme is not present, results in a poorly resolved enzyme fraction. However, the use of low ionic strength buffer in combination with a "pseudoattinity" type of agarose gel provides excellent resolution of enzyme from the majority of protein contaminants. The choice of the proper type of agarose gel is determined by establishing the molecular weight of eluted reductase through a comparison with protein standards of known molecular weight. Agarose gel in which the reductase elutes at an apparent molecular weight of less than 10,000, is suitable for proper enzyme purification. Unfortunately, gels of this type are difficult to obtain. Thus, an alternative fractionation (sucrose density gradient ultracentrifugation) has been developed to replace the BioGel filtration.

Method 227 Sucrose Density Gradient Ultracentrifugation. The 5 to 20% sucrose density gradient contains 50 mM potassium phosphate buffer, pH 7.0, 50 mM KC1, 30 mM EDTA, and 2 mM dithiothreitol (buffer D), in a total volume of 38 ml. The concentrated heat-treated enzyme pellet is dissolved in the above buffer at a sucrose concentration of 2% and a protein concentration of approximately 1.5 mg/ml. (It is important to apply a minimum of 500 units of enzyme activity to the gradient if a good recovery of enzyme activity is to be obtained. However, overloading of the gradient with more than 2000 units of enzyme activity will result in a poor purification of HMG-CoA reductase.) The ultracentrifugation is carried out at 14° for 41 hr at 25,000 rpm in a Beckman Model L3-50 ultracentrifuge, with a SW-27 swinging-bucket rotor. The gradient is then tapped at room temperature and collected in 35 fractions (each 1 ml) with an Isco Model 184 tube 27

D. A. Kleinsek and J. W. Porter, J. Biol. Chem. 254, 7591 (1979).

468

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[55]

holder and piercing mechanism. The fractions that contain the peak enzyme activity and minimal protein (normally tubes 16 to 20) are pooled. Enzyme Concentration. Before application to the affinity column it is necessary to concentrate the dilute enzyme solution from the BioGel filtration or sucrose density gradient ultracentrifugation steps. These preparations are concentrated in collodion bags at room temperature to a protein concentration of 1 mg/ml or greater. After concentration, the enzyme solution is dialyzed against the low ionic strength buffer used for affinity chromatography. The normal recovery of enzyme activity after concentrating and dialyzing is about 50%. Diluting or dialyzing the enzyme preparation before concentrating results in a greater loss of enzyme activity. A t~nity Chromatography. Affinity chromatography is carried out at 4° with a thioester-linked agarose-hexane-coenzyme A gel. The wet gel, 0.2 ml, is packed into a Pasteur pipette and then washed with approximately 20 volumes of a solution containing 25 mM potassium phosphate, pH 7.0, 1 mM EDTA, and 10% sucrose. After equilibration of the gel with this buffer, enzyme (1400 units) is rapidly cooled in ice to 4° and applied to the gel. A flow rate of 1 ml per l0 min is maintained during the binding process. One-milliliter eluate fractions are collected, and light absorbance at 280 nm is measured. The majority of protein, with negligible reductase activity, appears in the first two 1-ml fractions. When light absorbance decreases to the base-line level, reductase is eluted with binding buffer containing 0.5 M KCI. The elution flow rate is 1 ml/min, and fractions of 1 ml are collected. Of the applied enzyme, 95-100% is recovered in the first and second fractions. Yield, Purity and Properties of Enzyme (Methods 1 and 2)

Yield. The above procedure normally yields (from 25 rat livers) about 10% of the total enzyme activity present in the solubilized enzyme state, or 3-4% of that in microsomes. Extraction of enzyme by a repeated freeze-thawing of the microsomes increases this yield somewhat. About 2 ~g of pure enzyme are obtained from l0 g of rat liver. Thus, per l0 g of rat liver tissue, approximately 60 ~tg of active microsomal HMG-CoA reductase are available for purification. Therefore, large amounts of starting material are required for a substantial yield of enzyme. A typical purification by Methods 1 and 2 is summarized in Table I. The final specific activity is approximately 19,0(R)-20,000 units per milligram of protein when the BioGel agarose column is used. A slightly lower value of around 17,000 is obtained when sucrose density gradient ultracentrifugation is substituted. The overall purification-fold from microsomes varies between 3700 and 4100, and both methods yield homogeneous enzyme.

[55]

RAT LIVER H M G - C o A

469

.o

¢-q

z

REDUCTASE

>,

~ ~D

o

'1"

~

o

m gl

U2

oo

O 7 O

CO

O •~

cO

O ~ e., cO O 02

e~

cOr, e ~

470

HYDROXYMETHYLGLUTARYL-COA ENZYMES

[55]

Enzyme Purity. 24 The enzyme is judged to be homogeneous by a number of physicochemical criteria. The purified enzyme (10-40/zg) migrates as a single band of protein when electrophoresis is carried out on SDS-5% polyacrylamide disc gels. Furthermore, it migrates as a single band of protein (10-40/xg) with an R t o f 0.52 on 5% nondenaturing polyacrylamide disc gels when electrophoresed in a pH 8.9 system. This single band of protein comigrates with HMG-CoA reductase activity. Purified enzyme reacts against crude antiserum to form a single precipitin band on Ouchterlony double diffusion plates. Size. ~r The molecular weight of HMG-CoA reductase is 200,000, as determined by molecular sieve chromatography on two different gel matrices, agarose gel (BioGel) and dextran gel (Sephadex), and it consists of four subunits of 51,000 MW each, as determined by SDS-disc gel electrophoresis. Upon cold inactivation, the enzyme remains as a 200,000 MW entity. Thus, the molecular basis of cold inactivation is probably due to discrete conformational changes that affect the active site of HMG-CoA reductase. Apparent Km Values. 27Spectrophotometric assays of the initial velocity of oxidation of NADPH have been used to determine HMG-CoA reductase reaction rates. From double reciprocal plots of the data in which one substrate concentration was present in saturating amounts and the other concentration was varied, the apparent K m values were found to be 1.7 x 10-5 M for D-HMG-CoA and 3.0 × 10-5 M for NADPH. The saturating concentrations of DL-HMG-CoA and NADPH used were 3.6 × 10-4 M and 7.8 × 10-4 M, respectively. The concentration of DL-HMG-CoA was determined by light absorbance at 260 nm and by sulfhydryl analysis with Ellman reagent after complete hydrolysis of HMG-CoA with base. The concentration of NADPH was determined from light absorbance at 340 rim. Reversible CoM Lability. 2r Both soluble and microsomal HMG-CoA reductase exhibit reversible cold lability in low ionic strength phosphate buffer (0.1 M potassium phosphate, pH 7.0, 2 mM dithiothreitol). At low protein concentrations, partially purified enzyme (0.11 mg/ml) loses 50% of its activity within 90 sec. After 70% of the activity is lost, a slower rate of decline is observed until approximately 70 min, at which time a complete cessation of enzyme activity occurs. Rapid and complete recovery of enzyme activity occurs within 7.5 min when the solution is warmed at 37°. Microsomal enzyme exhibits a similar but slower loss at 4° and a rapid recovery of enzyme activity at 37° . pH and Temperature Optima. 27 The enzyme has a pH optimum of 6.25-7.25 and a temperature optimum in 0.1 M phosphate buffer, pH 7.0, and 2 mM dithiothreitol of approximately 47°.

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REDUCTASE

471

Isoelectric Point. 27 The enzyme has a pI of 6.2, as determined from isoelectric focusing on a 5% polyacrylamide disc gel. Stability and Storage. 2s Soluble enzyme, at a protein concentration of 1.2 mg/ml in buffer B, exhibits a significant loss of activity at room temperature, i.e., 25% and 50% after 5 and 19 hr, respectively. Slow freezing at - 20°, or fast freezing of the soluble enzyme in a Dry Ice-ethanol bath results in a 20% and 10% loss in activity, respectively. Heat-treated enzyme, at a concentration of 2 mg/ml, is stable at room temperature for 30 hr, and at 4°, only a 10% loss of activity of heat-treated enzyme occurs in 5 days. Slow or fast freezing and subsequent storage at - 2 0 ° for 5 days results in a negligible loss of enzyme activity. Activity of the purified enzyme is rapidly lost in low ionic strength buffer, but in the presence of the affinity eluent buffer the enzyme is stable at room temperature. Fast freezing of the enzyme results in variable losses of 10-30%, which are dependent on the protein concentration. Enzyme stored at 4° or - 2 0 ° is preincubated for 5-10 min at 37° before assays are carried out for enzyme activity. Both the soluble and microsomal enzyme require a sulfhydryl reagent for stability. Immunogenicity of the Enzyme. 24 Enzyme from the heat treatment step is effective in generating antiserum to HMG-CoA reductase. The enzyme is dispersed 1 : 1 with complete Freund's adjuvant for the initial injection, and 1 : 1 with incomplete Freund's adjuvant for booster injections. The titer of antibody is raised by subcutaneous multisite injections of the enzyme preparation into a rabbit. Additional injections are made at 2-week intervals, and blood is withdrawn and assayed for antibody titer at periodic intervals. Enzyme Orientation. 27 HMG-CoA reductase is solubilized from microsomal membranes by mild treatment, and it remains soluble in aqueous solution. The soluble enzyme and the membrane-bound microsomal enzyme have similar neutralization end points per unit of enzyme activity when titrated with antiserum produced against the soluble species. These physical and immunochemical properties of the enzyme support the conclusion that it is an extrinsic protein of the endoplasmic reticulum in which the active site of the enzyme is positioned toward the cytosol. This positioning is consistent with the location of HMG-CoA synthesis and mevalonate phosphorylation within the cell. Other Enzyme Fractionation Steps. 28DEAE-cellulose and NADP ÷ affinity chromatography also have been used successfully as fractionation steps, with respectable yields, in the purification of the enzyme. Both

2s D. A. Kleinsek, Ph.D. thesis, University of Wisconsin-Madison, 1979.

472

H Y D R O X Y M E T H Y L G L U T A R Y L - C o A ENZYMES

[55]

columns require a low ionic strength buffer for binding of the enzyme, and elution is effected by increasing the ionic strength of the buffer with KC1. Inactive H M G - C o A reductase. 24 The presence of an inactive species of HMG-CoA reductase which is formed in the liver or during the purification procedure and copurifies with the active species has been demonstrated by immunochemical and physical tests. On Ouchterlony double diffusion plates, the unbound protein fraction from the CoA affinity gel shows a partial identity of antigenic determinants to the active enzyme fraction that binds to, and is eluted from, the gel. Also, the majority of the protein in the unbound fraction shows an identical Rt with that of the purified active enzyme on SDS-polyacrylamide disc gel electrophoresis. Other HMG-CoA Reductase Purification Procedures This section describes purification procedures that employ two affinity chromatographic steps in conjunction with other purification techniques described in Method 1. These procedures also yield high specific activity enzyme (Table II). Method 3 z9 Preparation o f Microsomes a° and Solubilization o f the Enzyme. al Microsomes are obtained from the livers of male Sprague-Dawley rats weighing 200-300 g and fed ad libitum a 5% cholestyramine powdered rat chow for 4 days. The animals are sacrificed at the middle of the 12-hr dark period. Each rat liver is placed in 25 ml of ice-cold buffer A (40 mM potassium phosphate, 50 mM KC1, 30 mM potassium EDTA, and 0.1 M sucrose, pH 7.2). Homogenization of the liver is effected by six passes with a motordriven, tight-fitting glass-Teflon Potter-Elvehjem homogenizer. The homogenate is then subjected to two successive 15-min centrifugations at 10,000 g at 4°, and the postmitochondrial supernatant solution is centrifuged at I00,000 g for 60 min. The microsomal pellet obtained is resuspended in buffer A, and then centrifuged at 100,000 g for 45 min. The resulting pellet is resuspended in buffer A to a concentration of about 82 mg/ml, and solid dithiothreitol is added to a final concentration of 10 mM. Homogenization of this suspension is carried out with a hand-driven, all-glass Potter-Elvehjem hoqaogenizer (Kontes; clearance 0.004-0.006 inch), and the suspension is divided into 3-ml aliquots in glass tubes and ea P. A. Edwards, D. Lemongello, and A. M. Fogelman, Biochim. Biophys, Acta 574, 123 (1979). 3o p. A. Edwards and R. G. Gould, J. Biol. Chem. 247, 1520 (1972). alp. A. Edwards, D. Lemongello, and A. M. Fogelman, J. Lipid Res. 20, 40 (1979).

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TABLE II PURIFICATION OF HMG-CoA REDUCTASE FROM RAT LIVER BY METHODS 3, 4, AND 5

Ste~ Microsomal suspension

Soluble extract

35-50% (NI-I4)2SO4 30-50% (NI-Iq)2SO 4 35-50% (NH4)~SO4 Heat treatment

Blue Sepharose Afli-Gel Blue Agarose-CoA HMG-CoA agarose

Total protein (rag) 11,554c 5,179 a 3,202 e 932 291 307 330 79 54 84 20 2.93 3.95 2.13 0.790 0.164 0.193

Total activity (units)b 79,721 28,608 12,807 71,421 10,501 70,650 21,822 8,581 66,786 21,678 10,246 30,364 12,680 6,275 13,425 6,322 1,129

Specific activity (units/rag protein) 6.9 6.0 4.0 76 36 230 66 112 1,239 258 500 10,311 3,220 2,897 17,405 38,400 5,842

Yield (%)

Purification (fold)

100 100 100 90

1 1 1 11

82 89 76 67 84 76 80 38 44 49 17 22 9

9 33 12 30 180 46 127 1494 576 724 2522 6860 1461

a For each purification step, values are given in the order of Methods 3, 4, and 5. b The data in this table are normalized to a rat kill of 25. c Data derived from studies of Edwards e t al. 31 (Method 3) in which livers from 7 rats were used. a Data derived from studies of Ness e t al. 33 (Method 4) in which 14 rats were used. e Data derived from unpublished results ~ (Method 5) of studies in which 106 rats were used.

frozen at a rate of 6-8 ° per min. The microsomes can be stored at - 20 ° for up to 2 months. Solubilization of the reductase is initiated by thawing the microsomal suspension at room temperature or at 37 ° and then adding an equal volume of 50% glycerol in buffer B (buffer A plus 10 m M dithiothreitol) preheated to 37 ° . After homogenization of the suspension with ten downward passes of a hand-driven, aU-glass Potter-Elvehjem homogenizer, the suspension is incubated at 37 ° for 60 min. A threefold dilution with buffer B warmed to 37 ° is then made to yield a final glycerol concentration of 8.3%. The microsomal suspension is again homogenized with ten downward passes of the homogenizer pestle. After centrifugation at 100,000 g for 60 min at 25 °, the supernatant solution is removed and used immediately for enzyme purification. All subsequent steps of e n z y m e purification are performed at room temperature.

474

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[5S]

Preparation of Enzyme for Affinity Chromatography. The protein precipitating between 35 and 50% saturation with ammonium sulfate is dissolved in buffer B containing 30% glycerol (v/v) and 1.0 M KC1. Fourmilliliter aliquots of this solution, at a protein concentration of 3.6--4.8 mg/ml, are heated at 65° for 8 min and then centrifuged at 100,000 g for 30 min. The supernatant solution is diluted 1 : 1 with buffer B, and the enzyme is precipitated with ammonium sulfate (0 to 50% saturation). The solution is centrifuged and the pellet is dissolved in a small volume of buffer B and stored overnight at room temperature under nitrogen. Affinity Chromatography. Two types of affinity gel are used to purify HMG-CoA reductase. The first type, Blue Sepharose, contains the chromophore of Blue Dextran, Cibracron Blue F3GA, a sulfonated polyaromatic blue dye. It interacts with HMG-CoA reductase via the binding sites for both NADPH and (S)-HMG-CoA.29 The dye also interacts with proteins that possess a supersecondary structure called the dinucleotide fold. zz The enzyme suspension is incubated at 37° for 30 min and then centrifuged at 100,000 g for 30 min. Enzyme present in the supernatant solution, which contains less than 19.0 mg of protein, is applied to a Blue Sepharose affinity column, 5.5 × 1.0 cm. The gel is then eluted sequentially with two column volumes of buffer C (buffer A plus 2.5 mM dithiothreitol), which also contains the following sets of components, listed in the order of application: (a) 4 mM NAD ÷, 0.2 M KC1, (b) 4 mM NADH, 0.2M KC1; (c) 4 mM NADPH, 0.2M KCI; (d) 2 mM NADPH, 0.2M KCI, 0.75 mM CoA. The reductase is then eluted with 10 column volumes of buffer C plus 0.5 M KC1. The enzyme is concentrated and diluted to an ionic strength of 0.09 before application to an agarose-CoA column (0.7 × 1.3 cm). The gel is washed with 10 column volumes of buffer C, diluted 1 : 1 with a solution containing 0.1 M sucrose and 2.5 mM dithiothreitol, and then with 6 volumes of buffer C. The reductase is eluted with 3 column volumes of buffer C containing an additional 0.4 M KC1. Protein Determination. Soluble protein is determined by the method of Lowry et al. zn and by a modification of the Bradford procedure with Coomassie Brilliant Blue G-250. 3~ Enzyme Purity. The enzyme, specific activity of 17,500 units per milligram of protein, migrates as a single band of protein (15 ~g) on SDS and nondenaturing 5% polyacrylamide disc gel electrophoresis, and enzyme activity comigrates with the single band of protein. As determined by SDS-disc gel electrophoresis, the enzyme has a subunit molecular weight of 52,000. The high specific activity obtained for the Method 3 preparation 32 S. T. Thompson, K. H. Cass, and E. Stellwagen, Proc. Natl. Acad. Sci. U.S.A. 72, 669 (1975).

[55]

RAT LIVER H M G - C o A

REDUCTASE

475

is similar to that for the enzyme demonstrated to be homogeneous by Method 1. However, when antisera were raised to four preparations of enzyme varying in specific activity from 15,000 to 22,500, Ouchterlony double diffusion patterns against impure enzyme preparations yielded three precipitin lines.

Method 4 ~3 Preparation of Microsomes and Solubilization of Enzyme. Male Sprague-Dawley rats, weighing 125-150 g at time of purchase, are fed a 2% cholestyramine powdered Wayne Lab Blox diet for 6-8 days prior to sacrifice. The animals are then decapitated at 3 hr into the 12-hr dark period, and the livers are placed in cold PESK buffer (40 mM potassium phosphate, 30 mM EDTA, 0.1 M sucrose, 50 mM KCI, and 1 mM dithiothreitol, pH 7.2). The livers are minced with scissors and then homogenized with 4 volumes of cold PESK buffer with a motor-driven Potter-Elvehjem glass-Teflon homogenizer. The homogenate is centrifuged at 10,500 g for 15 min, and the postmitochondrial supernatant solution is centrifuged at 99,500 g for 60 min. The microsomal pellets are frozen and stored at - 2 0 ° for up to 10 days before solubilization of the enzyme. The solubilization of the enzyme is based on the method of Heller and Gould. ~8 The frozen microsomal pellets are thawed at room temperature and then homogenized in cold PESK buffer (12 ml per each microsomal pellet). Each pellet is derived from 7 g of liver. The homogenate is centrifuged at 100,000g for 60 min at 4°, and the supernatant solution containing the soluble enzyme activity is extracted with a Pasteur pipette and saved. The microsomal pellet is frozen, thawed, and extracted four additional times as stated above. The five extracts are combined and used for further enzyme purification. Preparation of Enzyme for A~nity Chromatography. The extract is incubated at 37° for 90 min and then centrifuged at 20,000 g for 10 min. The supernatant solution is fractionated between 30 and 50% saturation with ammonium sulfate, and the precipitate is removed by centrifugation. The sedimented pellet is dissolved in PESK buffer and frozen at - 2 0 °. The concentrated extract is then thawed at 37°, and glycerol and potassium chloride are added to the extract to final concentrations of 33% and 1 M, respectively. This enzyme solution is added to prewarmed tubes and heated at 64° for 10 min. The turbid solution is rapidly cooled to room temperature by placement in an ice-water bath and then centrifuged at 33 G. C. Ness, C. D. Spindler, and M. H. Moffier, Arch. Biochem. Biophys. 197, 493 (1979).

476

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[55]

144,000 g for 15 min. The supernatant solution containing the enzyme activity is saved for subsequent affinity chromatography. A~nity Chromatography. The first affinity chromatographic step utilizes an Affi-Gel Blue column (1.5 x 4.0 cm) containing the ligand Cibacron Blue F3GA. The column is equilibrated at room temperature with PESK buffer containing 0.2 M KCI, and the enzyme solution is diluted fivefold with PESK buffer to reduce the concentration of KC1 to 0.2 M. After approximately 4800 units of heat-treated enzyme in a volume of 300 ml is applied, the column is washed with 100 ml of PESK buffer containing 0.2 M KCI. The enzyme is then eluted in PESK buffer with a linear gradient of 0.2 to 2.0 M KCI. Fractions of 7.5 ml are collected, and those containing more than 100 units of activity are saved and concentrated with an Amicon ultrafiltration cell containing a YM-10 filter. Repeated ultrafiltration with additions of PESK buffer removes the KC1. Glycerol is then added to a final concentration of 50%, and the enzyme is stored at - 70°. The final step of purification of the enzyme requires a thioester-linked agarose-hexane-HMG-CoA column (0.6 × 2.0 cm). The enzyme is thawed and water is added to dilute the glycerol concentration to 25%. Thirty-eight milliliters of an enzyme solution containing 2050 units are applied to a column which has been equilibrated at room temperature with buffer A (PESK : glycerol : water mixed in ratios of 1 : 1 : 2). The column is then eluted sequentially with the following solutions, and 4-ml fractions are collected: 40 ml of buffer A containing 70 mM KCI; 10 ml of buffer A containing 70 mM KCI and 200/xM RS-HMG-CoA; and buffer A containing 0.5 M KC1. Most of the HMG-CoA reductase is eluted by the addition of HMG-CoA to buffer A. Protein Determination. The protein concentrations of soluble fractions are determined by the Coomassie dye-binding method, a4 Enzyme Purity. Small amounts of protein are analyzed by SDS and nondenaturing polyacrylamide disc gel electrophoresis. Purified enzyme (14/zg) migrates as a single band corresponding to a molecular weight of 50,000 on SDS-7.5% polyacrylamide disc gel electrophoresis. On native 6% polyacrylamide disc gels, electrophoresis of 8/zg of protein yields a single staining band, which comigrates with enzyme activity. The specific activity of purified enzyme varies from 32,000 to 46,000 nmol of NADPH oxidized per minute per milligram of protein. However, when protein determinations were carried out by the Lowry method 26the specific activity value of 46,800 decreased to 27,000. This value is approximately 40% higher than the value reported for the homogeneous enzyme preparation obtained by Method 1. a4 M. M. Bradford, Anal. Biochern. 72, 248 (1976).

[55]

RAT LIVER H M G - C o A

REDUCTASE

477

Method 5 ~ Preparation of Microsomes and Solubilization of the Enzyme. The treatment of animals and the preparation of microsomes are carried out as described for Method 1, but microsomal pellets are stored at - 6 0 °. The solubilization of the reductase is performed by thawing the microsomes at room temperature and then resuspending the pellets at a protein concentration of 70-85 mg/ml. This procedure is similar to that of Method 3, except that the solubilization buffer is at pH 7.0 and the homogenization is carried out with a motor-driven Teflon pestle in a Potter-Elvehjem type glass homogenizer. Preparation of Enzyme for AJ~nity Chromatography. The salt fractionation of enzyme is performed with a saturated solution of enzyme grade ammonium sulfate, pH 7.0, as described in Method 1. The protein pellet derived from this step is dissolved in buffer containing 50 mM potassium phosphate, pH 7.0, 30 mM EDTA, 1.0 M KCI, 30% (v/v) glycerol, and 10 mM dithiothreitol, and the heat treatment is carried out at 65° for 8 min as described for Method 1. A~nity Chromatography. Purification of the reductase with Affi-Gel Blue and by HMG-CoA affinity chromatography is accomplished by a minor modification of the procedure of Ness et al. az Heat-treated enzyme is diluted 1 : 1 with PESK buffer and precipitated between 0 and 50% saturation with ammonium sulfate as described in Method 1. The pellet is dissolved in a minimum amount of buffer containing 50 mM potassium phosphate, pH 7.0, 2 mM dithiothreitol, 30 mM EDTA, and 10% (v/v) glycerol, and dialyzed for 1 hr. After dialysis, solid dithiothreitol is added to the enzyme solution to give a final concentration of 5 mM. The Affi-Gel Blue column (1.2 × 5.0 cm) is then equilibrated with buffer A [50 mM potassium phosphate, pH 7.0, 30 mM EDTA, 10% glycerol (v/v), and 5 mM dithiothreitol]. Enzyme (approximately 22,000 units) is applied to the column at a flow rate of 1 ml per 2 min. Protein on the column is eluted sequentially with buffer A and buffer A plus 0.5 M KCI. Enzyme is eluted at a flow rate of 1 ml/min with buffer A containing 2.0 M KCI, and eluates are collected in 2-ml fractions. Those tubes with more than 300 units of enzyme activity are pooled and stored overnight at room temperature, when necessary. The thioester-linked agarose-hexane-HMG-CoA column (0.6 x 1.8 cm) is equilibrated with buffer B [50 mM potassium phosphate, pH 7.0, 2 mM dithiothreitol, 30 mM EDTA, and 15% (v/v) glycerol]. Enzyme is dialyzed against the above buffer, and 10,543 units of activity are applied to the affinity column at a flow rate of 1 ml in 8-10 min. The column is 3s T. A. Baker, R. E. Dugan, and J. W. Porter, unpublished results.

478

HYDROXYMETHYLGLUTARYL-CoA ENZYMES

[55]

sequentially washed with buffer B, buffer B containing 70 mM KCI, buffer B containing 70 mM KC1 and 0.2 mM DL-HMG-CoA, and buffer B containing 1.0 M KCI. Enzyme is eluted at the fastest flow rate possible with buffer B containing 70 mM KCI and 0.2 mM DL-HMG-CoA, and eluates are collected in l-ml fractions. The fractions containing enzyme activity are pooled, and KCI is added to a final concentration of 2.0 M before assaying for total enzyme activity. Glycerol is added to a final concentration of 50% before storage at - 6 0 ° . Protein Determination. Protein concentrations of soluble enzyme preparations are determined by the method of Lowry et al. 26 Enzyme Purity. The purified enzyme has a specific activity of approximately 5800 nmol of NADPH oxidized per minute per milligram of protein. However, 40/zg of the enzyme migrates as one major band corresponding to a molecular weight of 50,000 on SDS-10% polyacrylamide disc gel electrophoresis. Comparison of Methods A comparison of the five methods of purification of HMG-CoA reductase from microsomes shows that each includes five major fractionation steps and that there are many similarities in these methods. All methods incorporate a freeze-thaw treatment of microsomes to solubilize enzyme. All the methods also include an ammonium sulfate fractionation, a heat treatment step, and at least one specific affinity chromatographic separation. The major differences among these methods are the following. Methods 1 and 2 use a freeze-thaw treatment of microsomes to solubilize enzyme, whereas Method 4 uses a repeated freeze-thaw extraction procedure. Methods 3 and 5 solubilize enzyme by incubation with glycerol after a freeze-thaw treatment of microsomes. Methods 3, 4, and 5 substitute the affinity gels Affi-Gel Blue or Blue Sepharose in place of the BioGel filtration or sucrose density gradient ultracentrifugation step presented in Methods 1 and 2, respectively. Methods 4 and 5 use an agarose-hexane-HMG-CoA affinity gel as the final step of purification, whereas Methods 1, 2, and 3 use an agarose-hexane-CoA gel for the affinity chromatographic separation. Methods 3 and 4 show a recovery of enzyme units in the 17-22% range, which is greater than the 9% yield for Method 5 or the 3-4% yield for Methods 1 and 2. The increased yield of enzyme obtained in Methods 3, 4, and 5 results from an increase in the amount of enzyme solubilized from the microsomes. In Methods 3, 4, and 5, yields of 90, 76, and 82%, respectively, are observed, as compared to 31% for Methods 1 and 2.

[55]

RAT LIVER H M G - C o A REDUCTASE

479

Additional modest gains are also observed in the heat treatment or ammonium sulfate fractionation steps for Methods 3, 4, and 5. The amount of protein recovered for Methods 1 and 2 is 53 /xg, whereas Methods 3, 4, and 5 yield 790, 164, and 193 /zg, respectively. These figures are based on a normalized rat kill of 25, and they appear to correlate with the final enzyme activity yield, except for Method 3 where much larger amounts of protein are obtained. The increase in protein by this method appears to be due to a three- to fivefold increase in the amount of starting enzyme units over that in other methods. This high microsomal activity may be a result of the treatment of the animals (i.e., 5% instead of 2% cholestyramine in the diet) or to the preincubation of microsomes before solubilization of the enzyme. The degree of purification for enzyme derived from microsomes were 4100, 3700, 6900, 2500, and 1500, respectively, for Methods 1-5. Methods 1 and 2 produce enzyme of high specific activity (17,000 to 19,000 units per milligram of protein). Enzyme obtained by these methods has undergone a number of stringent physicochemical tests for homogeneity. By all the criteria used (including electrophoresis of a range of protein concentrations on SDS and native polyacrylamide disc gels and immunochemical experiments with several levels of protein) the enzyme is judged to be homogeneous. Small amounts of enzyme from Method 3 migrate as a single band on SDS and nondenaturing polyacrylamide disc gel electrophoresis. Although the specific activity is similar to enzyme purified by Method 1, Method 3 produces enzyme that is immunochemically impure, and thus it would appear not to be homogeneous. Method 4 yields enzyme of a specific activity greater than other reported values. Small amounts of this protein act as a homogeneous species on SDS and native polyacrylamide disc gel electrophoresis. Method 5 produces enzyme of lower specific activity than that obtained by Methods 1 through 4. However, large amounts of this enzyme migrate as a single band of protein that corresponds with the subunit molecular weight of HMG-CoA reductase when subjected to SDS-polyacrylamide disc gel electrophoresis. In addition, rocket immunoelectrophoresis of a crude HMG-CoA reductase preparation into antibody that was produced in response to the purified enzyme showed one major and one trace precipitin arc. Acknowledgments This work was supported in part by a Research Grant, H L 16364, from the National Heart and Lung Institute,National Institutesof Health, United Stales Public Health Service, and by the Medical Research Service of the Veterans Administration.