123

Biochimica et Biophysics Acta, 574 (1979) @ Elsevier/North-Holland Biomedical Press

123-135

BBA 57394

PURIFICATION AND PROPERTIES 3-HYDROXY-3-METHYLGLUTARYL

PETER

A. EDWARDS,

DONNA

LEMONGELLO

Division of Cardiology, Department Los Angeles, CA 90024 (U.S.A.) (Received

November

OF RAT LIVER COENZYME A REDUCTASE

of Medicine,

and ALAN M. FOGELMAN University of California Los Angeles,

3rd, 1978)

Key words: Hydroxymethylglutaryl-CoA

reductase;

(Purification, Properties, Rat liver)

Summary

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity corn&rated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200 000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52 000 + 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11 303 and 19 584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment. Introduction The regulation of 3-hydroxy-3-methylglutaryl 1.1.1.34) has been under intensive investigation

coenzyme A reductase in recent years because

(EC it is

124

generally considered to be the rate-determining enzyme for the conversion of acetate to cholesterol [ 1,2]. A detailed study of the physiological regulation of the reductase is dependent upon the availability of monospecific antibody to this enzyme, which in turn is dependent on the availability of substantial quantities of the purified enzyme. A number of investigators have reported purification of the enzyme from the microsomal fraction of rat [ 3-81 or chicken [9] liver. However, the specific activities of the purified enzyme from untreated rats varied from 3 to 691 nmol mevalonate formed/min per mg protein [3,4,6-S]. In rats fed cholestyramine, a resin which removes bile acids from the enterohepatic circulation [lo] and stimulates cholesterol biosynthesis [ 111, the specific activity of the purified enzyme is reported to vary from 2524 [7] to 9500 [5] nmol mevalonate formed/min per mg protein. The reasons for these large variations in specific activity have yet to be determined. We report here a rapid method of purifying rat liver 3-hydroxy-3-methylglutaryl CoA reductase to apparent homogeneity on Blue Sepharose and Agarose-CoA affinity columns. The purified enzyme was obtained in significantly higher yields than previously reported [5] for enzyme of comparable specific activity. In this communication we have investigated, using immunochemical methods, whether the inactivation of the enzyme following mevalonolactone treatment [12] results in an inactive form of the enzyme which is capable of cross-reacting with anti-reductase antibody. We have also investigated the effect of cholestyramine feeding on the reductase levels. Materials and Methods Materials. NAD, NADH, NADP, NADPH, CoASH and dithiothreitol were obtained from Sigma; Agarose-hexane-CoA (Type 5) from P.L. Biochemicals; Blue Sepharose CL-6B from Pharmacia. The free chromophore Cibacron Blue F3GA was a generous gift from Dr. E. Stellwagen. Cholestyramine (Questran) was obtained from Mead Johnson; Agarose 0.5 m, SDS and acrylamide from Bio-Rad and Ouchterlony plates from Hyland. Animals. Rats were housed under a reverse illumination cycle [ 131, had free access to food and water, and were fed powdered rat food supplemented with 5% cholestyramine for 4 days before they were killed. Where indicated, animals were fed a normal diet, free of cholestyramine. Solubilization and assay of the reductase. Unless otherwise stated, animals were killed by decapitation at the middle of the 12 h dark period. Livers were removed and homogenized in 25 ml buffer A (0.1 M sucrose, 0.05 M KC1 0.04 M potassium phosphate, 0.03 M EDTA, pH 7.2) [133. The preparation of the microsomal fraction, the solubilization of the enzyme from the membranes and the assay of the reductase in either the microsomal fraction or after solubilization have been described [ 131. One enzyme unit is defined as the oxidation of 1 nmol NADPH/min and is equivalent to the synthesis of 0.5 nmol mevalonatelmin. Purification of hydroxymethylglutaryl CoA reductase. All subsequent steps of enzyme purification, after solubilization of the enzyme [ 131, were performed at room temperature. The protein precipitating between 35% and 50% ammonium sulfate was dissolved in buffer B (buffer A plus 10 mM dithiothrei-

125

tol) containing 30% glycerol (v/v) and 1.0 M KC1 using a minor modification of the method of Kleinsek et al. [5]. 4-ml aliquots of the solution (3.6-4.8 mg protein/ml) were heated for 8 min at 65°C. After centrifugation at 100 000 X g for 30 min the supernatant was removed, diluted with an equal volume of buffer B and the protein precipitating between 0 and 50% saturation with ammonium sulfate collected by centrifugation. This pellet, containing 69 + 13% (n = 9) of the original solubilized enzyme activity, was dissolved in a small volume of buffer B and stored overnight at room temperature under nitrogen. No loss of enzyme activity was observed after storage under these conditions. Overnight storage at 4°C resulted in a loss of approximately 8% of the enzyme activity. The solution was warmed at 37°C for 30 min, centrifuged at 100 000 Xg for 30 min and the supernatant containing all the enzyme activity and less than 19.0 mg protein was applied to a Blue Sepharose affinity column (5.5 X 1.0 cm). The column was washed sequentially with two column ~01s. of buffer C (buffer A plus 2.5 mM dithiothreitol) containing 4 mM NAD plus 0.2 M KCl, 4 mM NADH plus 0.2 M KCl, 4 mM NADPH plus 0.2 M KCl, 2 mM NADPH plus 0.2 M KC1 and 0.75 mM CoASH. The reductase was then eluted in ten column ~01s. of buffer C containing an additional 0.5 mol KCl/l. The enzyme solution was concentrated, diluted to an ionic strength of 0.09 and applied to an Agarose-CoA (type 5) affinity column (0.7 X 1.3 cm) and the column washed with ten column ~01s. of buffer C diluted 1 : 1 with 0.1 M sucrose/2.5 mM dithiothreitol followed by six column ~01s. of buffer C. The purified enzyme was eluted in three column ~01s. of buffer C containing an additional 0.4 mol KCl/l. The use of Agarose-CoA affinity chromatography to purify the reductase was first reported by Beg and Brewer [14] and more recently by Kleinsek et al. [51. Polyacrylamide gel electrophoresis. Disc gel electrophoresis of the enzyme was carried out using gel system No. 6 of Maurer [15] except that the polyacrylamide concentration was 5%. The procedure of Weber and Osborn [16] was used for SDS-polyacrylamide gel electrophoresis. Samples were heated at 100°C for 4 min with 1% SDS and 1% fl-mercaptoethanol before electrophoresis on 5% acrylamide gels. Native gels were stained for at least 12 h with 0.01% Coomassie Blue G-250 in 12.5% trichloroacetic acid. SDS gels were stained for at least 1 h with Coomassie Blue R-250. Analysis of polyacrylamide gels for reductase activity. Before samples were applied, the gels were removed from the glass tubes and dialyzed against three changes of the gel buffer [15] diluted eight fold with water and containing 25 mM fl-mercaptoethanol. The gels were then sucked up into clean tubes and the protein sample applied and electrophoresed in the normal manner. The method of ‘popping’ the gels to dialyze out potential inhibitors has been described by Weiner et al. [ 171. After electrophoresis of the reductase the gel was sliced into 0.4 cm lengths and homogenized in a buffer containing 0.2 M KCl, 0.16 M potassium phosphate, 0.004 M EDTA, 0.01 M dithiothreitol, 0.2 mM NADPH, 0.002 M NADP, 0.01 M glucose 6-phosphate and 2.2 I.U. glucoseB-phosphate dehydrogenase. The assay was started by addition of 150 nmol

126

hydroxymethyl[3-‘4C]glutaryl CoA to the 0.5 ml assay. After 10 min, the assay was stopped by addition of KOH and the 14C content of the mevalonate determined as previously described for the assay of microsomal reductase [ 131. Protein determinations. The protein content of microsomal suspensions was determined by the biuret method [ 181. Soluble proteins were determined both by the method of Lowry et al. [19] and with Coomassie brilliant blue G-250 by a minor modification [13] of the method of Bradford [20]. Bovine serum albumin was used for a standard in all protein determinations. The protein concentration of three different preparations of purified reductase were determined by both methods and no substantial differences were observed. Preparation of anti-reductase antibodies. Four different preparations of reductase were used to prepare antibodies in two rabbits. The purity of each enzyme preparation was checked by SDS-polyacrylamide gel electrophoresis. The specific activities of the four preparations varied from 15 000 to 22 500 nmol NADPH oxidized/mm per mg protein. Rabbits were injected subcutaneously and intradermally with the reductase (0.02 mg), prepared as a stable emulsion in an equal volume of Freunds complete adjuvant. Rabbits were injected on three separate occasions during the subsequent 11 weeks with protein in either buffer or in Freunds incomplete adjuvant. Each rabbit received a total of 0.22 mg protein. Rabbits were bled from the ear 7-10 days after administration of antigen. The clotted blood was centrifuged and the serum removed. Crude y-globulin was obtained from the serum by three successive (NH,),SO, precipitations and the final precipitate was resuspended in one-half the original volume of serum and dialyzed extensively against borate-buffered saline [21]. The antibody was evaluated by immunodiffusion and immunoelectrophoresis [al]. Immunotitrations of solubilized reductase employed a constant amount of enzyme activity and increasing amounts of y-globulin in a total volume of 200 ~1. The mixture were incubated at 37°C for 30 min in 400~~1 plastic microfuge tubes before centrifugation for 10 min at 10 000 X g. The reductase activity in 150 r.rl of the supernatant was determined spectropnotometrically. Results Purification of hydroxymethylglutaryl CoA reductase The enzyme was purified to apparent homogeneity from microsomes obtained from animals fed a standard diet (Table I) or a diet containing 5% cholestyramine (Table II). The specific activity of enzyme purified from rats fed a normal diet or a diet supplemented with cholestyramine was 11 303 (n = 2) and 19 584 rt 1511 (n = 9) nmol NADPH oxidized/min per mg protein, respectively (Tables I-III). The purification of the reductase was dependent on the unique interaction between the enzyme and Cibacron Blue. Stellwagen and collaborators [2224] have demonstrated that enzymes containing the dinucleotide fold form a complex with blue dextran or its chromophore Cibacron Blue F3GA. As expected, the free chromophore Cibacron Blue F3GA, was a competitive inhibitor (with respect to NADPH) of the reductase with a Ki of 1.1 fl (Fig. 1A). However, the chromophore was a competitive inhibitor with respect

127 TABLE

I

PURIFICATION

OF HYDROXYMETHYLGLUTARYL

9 rats fed the standard specific

activities

Purification

diet were killed

at midnight

are given as nmol NADPH

step

Total protein

CoA REDUCTASE and the reductase

oxidized/min

FROM

purified

NORMAL

TABLE

3 235 333 106 16.83 0.50 0.060

Total units

Specific

activity

4 530 4 349 2 995 3 218 1938 696

1.4 13.05 28.19 194.80 3 838 11660

Yield

(%)

Purification (fold) 1 9 20 139 2 741 8 329

100 96 66 12 43 15

II

PURIFICATION OF AMINE-FED RATS

HYDROXYMETHYLGLUTARYL

CoA

REDUCTASE

FROM

CHOLESTYR-

7 rats fed 5% cholestyramine for four days were killed at midnight and the reductase purified microsomes. The specific activities are given as nmol NADPH oxidized/min per mg protein. Purification

step

Total protein

Total units

Specific

22 19 19 18 8 3

6.9 76.6 230 1239 10 311 17 405

activity

Microsomal suspension Soluble extract 35-50% (NH4)2S04 65°C. 8 min Blue Sepharose Agarose-CoA

3 235 261 86 15.09 0.82 0.22

322 998 782 700 502 759

from

the

Yield (W)

Purification (fold)

100 90 89 84 38 17

1 11.1 33.3 180 1494 2 522

(mg)

TABLE

The

per mg protein.

(mg) Microsomal suspension Soluble extract 3%50% (NH4)?SO4 65°C. 8 min Blue Sepharose Agarose-CoA

RATS

from the microsomes,

III

SPECIFIC ACTIVITY OF HYDROXYMETHYLGLUTARYL RATS AFTER VARIOUS TREATMENTS

CoA

REDUCTASE

PURIFIED

FROM

In most experiments a sample was electrophoresed on SDS-polyacrylamide gel electrophoresis and gave one major band. Km values were determined by the method of Eisenthal and Cornish-Bowden [311. Similar values were obtained from Lineweaver-Burke plots [131. (S)-HMG-CoA, (S)-hydroxymethylgiutarylCoA. Diet

normal 5% cholestyramine

Reductase spec. act. (nmol NADPH oxidized/ min per mg protein

Km (MM) NADPH

(S)-HMG-CoA

11660 10 946 29 225 22 561 14 267 15000 17 405 19 100 19 608 21600 17 493

75 56

2.0 1.8

51

2.6

87

1.85

128

, 0

10

20

‘INADPH

30

0

40

‘/RS-HMG-CoA

( mM I-’

,

20

I

I

40

60

(PM)-’

Fig. 1. Inhibition of hydroxymethylglutaryl CoA reductase by Cibacron Blue FBGA. Solubilized reductase was heated at 55’C for 15 min. centrifuged and the supernatant applied to a Blue Sepharose affinity column. The enzyme. eluted in buffer C containing 0.5 M KC1 and with a specific activity of 1100 units/ mg protein was used in the inhibition studies in the absence (0) or in the presence of Cibacron Blue F3GA (CB FBGA) at 0.655 PM (“) or 1.31 I.IM (A). In (B) the concentration of NADPH was 0.2 mM and in (A) (RS)-hydroxymethylglutaryl CoA was 300 /LM. Ki values were obtained by replotting the reciprocal of the intercept on the l/S axis vs. inhibitor concentration (inset to (A) and (B)).

to the substrate hydroxymethylglutaryl CoA with a Ki of 0.2 PM (Fig. 1B). These results suggested that the reductase might be bound to Blue Sepharose by interaction of the affinity ligand with the binding sites for both reductase substrates, NADPH and hydroxymethylglutaryl CoA. Support for this proposal came from experiments in which we were unable to elute significant amounts of enzymically active reductase from non-saturated Blue Sepharose affinity columns with 10 mM NADPH in the absence or presence of CoASH. However, approximately 75% of the bound enzyme was eluted in buffer C containing an additional 0.5 mol KCl/l. In order to purify the reductase to homogeneity we made use of the finding that many proteins that bind to affinity columns containing Cibacron Blue F3GA, are eluted in buffer of low ionic strength containing low concentrations (l-10 mM) of nucleotides [22]. When the Cibacron Blue affinity column was washed with various nucleotides the specific activity of the eluted enzyme was approximately 10 000 units/mg protein. Omission of the nucleotides resulted in enzyme specific activities of approximately 3000 units/mg protein. Kinetic properties

There were no significant differences in the Km values for either substrate for enzyme purified from normal or cholestyramine-fed rats; the mean Km for (S)hydroxymethylglutaryl CoA was approximately 2.0 PM and for NADPH approximately 67 DM (Table III). Evidence

for homogeneity

of hydroxymethylglutaryl

CoA reductase

The purified reductase gave one major band on disc polyacrylamide gel electrophoresis (Fig. 2A). The reductase activity comigrated with the protein

129

Blue

Front

Fig. 2. The reductase was purified from cholestyramine-fed animals to a specific activitY of 17 493 Units/ mg protein. Enzyme (15 pg) was electrophoresed on either gel No. 6 from Maurer [151 (A) 01 on 5% acrylamide gels in the presence of SDS (B).

band in samples prepared from both normal (Edwards, P.A., Lemongello, 3. and Fogelman, A.M., unpublished results) and cholestyramine-fed rats (Fig. 3). One major band was obtained when enzyme purified from both normal and cholestyramine-fed rats was electrophoresed on SDS-polyacrylamide gel electrophoresis (Fig. 2B). The molecular weight of the enzyme subunit was determined for six different purifications and estimated to be 51 800 + 2200. The molecular weight of the reductase purified through the Blue Sepharose affinity column and chromatographed in Sephadex G-200 was approximately 200 000. Immunochemical

Ouchterlony

studies

double-immunodiffusion

analysis

using antiserum

prepared

in

130

GEL SLiCE Fig. 3. The reductase was purified from animals fed the cholestyramine diet to a final specific activity of 17 493 unitslmg protein. Samples containing 2.8 j&gand 7.1 pg protein were electrophoresed on 5% acrylamide gels. One gel containing 7.1 pg protein was stained for protein and the second gel was sliced into 4-mm segments and assayed for 10 min for reductase activity. The position of the bromophenol blue is shown(F). Fig. 4. Ouchterlony double-diffusion patterns of the reductase. The center well contained 20 pg of antisera prepared against purified reductase. Well A, purified reductase, 1.13 @g and 24.5 units; well B, O-50% (NHq)zSOa fraction of crude solubilized enzyme. 20 units; well C. 35-50% (NHq)zS04 fraction. 94 bug and 13.5 units; well D, soluble enzyme fraction obtained after partial purification by (NHq)zS04 precipitation and heating 65’C for 8 min. 21.3 pg and 19.3 units: well E. the enzyme fraction in well D was further purified by precipitation with 50% (NHq)zS04, 23.7 fig and 26.9 units. The plates were developed for 1 day at room temperature and for 10 days at 4’C.

rabbits to purified reductase gave a single precipitin line when tested against purified protein (Fig. 4). Three precipitin lines were observed against impure enzyme preparations (Fig. 4). Immunoelectrophoresis using purified reductase showed one precipitin line.

Immunotitration

studies

We have previously demonstrated that administration of mevalonolactone to rats fed a normal diet resulted in a rapid decrease in microsomal enzyme activity [ 121. It was not known whether the loss in activity resulted from enzyme inactivation or increased degradation of the enzyme. When rats were fed a diet containing 5% cholestyramine and killed at midnight the specific activity of the microsomal enzyme was increased compared to control animals fed a normal diet (Table IV). Administration of increasing amounts of mevalonolactone to the rats 45 mm before they were killed resulted in a dose-dependent decrease in microsomal reductase specific activity (Table IV). Immunotitration of the reductase solubilized from the microsomes showed that the equivalence point was affected both by cholestyramine and by mevalonolactone administration (Table IV; Fig. 5); as the dose of mevalonolactone increased, decreasing amounts of enzyme activity were

131

TABLE EFFECT

IV OF DIET

ON EQUIVALENCE

POINT

OF SOLUBILIZED

HYDROXYMETHYLGLUTARYL

CoA REDUCTASE Rats were treated and immunotitrations

carried out as described

in the legend to Fig. 5. The immunotitra-

tion data are given as the number of nmol NADPH oxidized/min which are inactivated per 1.0 ~1 antibody. The numbers in parenthesis refer to the number of assays from different animals. MVA. mevalonate. Diet

Mevalonate dose (mg/ZOO g body weight)

Spec. act. of microsomal reductase (nM MVA/min per mg protein)

Equivalence point (units inactivated/n1 antibody (’ S.E.))

50 100 150 200

0.45-0.80 3.5 -6.9 1.85 1.75 1.39 1.18

0.64 0.90 0.88 0.85 0.74 0.71

Normal 5% cholestyramine

* Significantly

different

from controls

by a two-tailed

(‘7) (7)

non-paired

Student’s

* 0.03 ? 0.03

*

/-test (P < 0.001).

inactivated per yl antiserum. The data are consistent with the presence, in the soluble fraction obtained from the microsomes, of an inactive species of hydroxymethylglutaryl CoA reductase which is capable of forming an antigenantibody complex. The data are also consistent with a direct correlation between the concentration of the inactive enzyme and the dose of mevalonolactone. The immunotitration data also demonstrate that the equivalence points for enzyme solubilized from rats fed a normal diet or a diet containing 5% cholestyramine for four days were significantly different (P < 0.001); 0.64 vs. 0.90 14

6

0

4

6 ANTIBODY

12

16

ADDED

20

24

(PI 1

Fig. 5. Immunotitration of the reductase. Animals were fed a normal diet (u), 5% cholestyramine for four days (.l or 5% cholestyramine for four days and then given 200 mg mevalonolactone/200 g body weight 45 mm before they were killed (sl. All animals were killed at the middle of the 12 h dark period. Enzyme solubilised from animals fed a normal diet was concentrated by precipitation with 50% (NH4)2S04 and resuspended in a small volume of buffer. (NH4)+04 precipitation of enzyme solubilized from cholestyramine-fed rats did not affect the immunotitration. The number of enzyme units added originally to each 200 ctl assay were 13.7 (~‘1, 13.7 (a), 15 (Al. Similar results were obtained in other experiments in which the amount of antibody was kept constant and the number of enzyme units added to each 0.2 ml incubation was varied. However, under these latter conditions a significant percentage of enzyme units were inactivated in the absence of antibody as a result of the instability of the reductase under dilute condi-

tions (Edwards,

P.A., Lemongello,

D. and Fagelman,

A.M., unpublished

results).

132

enzyme units were inactivated/d antibody, respectively (Table IV; Fig. 5). These data indicate that cholestyramine feeding results in a 40% activation of the enzyme. The antigen-antibody complex which precipitated after centrifugation had zero reductase activity. Preincubation of microsomes with excess antibody before addition of hydroxymethyl[ 3-‘4C]glutaryl CoA resulted in inhibition of at least 91% of the enzyme activity. Preliminary immunotitration studies with intact microsomes indicate that the equivalence points for the microsomal bound enzyme and for solubilized reductase were similar (Edwards, P.A.: Lemongello, D. and Fogelman, A.M., unpublished results). Discussion The purification of rat hepatic hydroxymethylglutaryl CoA reductase reproducibly yielded enzyme of high specific activity with recoveries of approximately 15-30% of the enzyme activity originally assayed in the microsomal fraction. These recoveries are 5-10 fold higher than those reported previously [5] for enzyme of comparable specific activity. The mean specific activities were 19 584 (n = 9) nmol NADPH oxidized/min per mg protein for animals fed diets containing 5% cholestyramine and 11 303 nmol NADPH oxidized/min per mg protein for animals fed a normal diet and killed at the time of maximum reductase activity (Table III). These values correspond to an 8300 and 3200 fold purification of the microsomal enzyme from normal and cholestyramine-fed rats, respectively. The specific activity of the purified enzyme obtained from rats fed a normal diet (11 303 units/mg protein) is an order to magnitude higher than the corresponding values of 6-1382 reported in the literature [3,4,6-S]. The value of 19 584 obtained from rats fed cholestyramine is similar to that recently by reported Kleinsek et al. [5], but significantly higher than the value of 5048 reported by Srikantaiah et al. [ 71. We have previously purified the reductase [ 131 by the method of Kleinsek et al. [5]. In our hands the latter method appeared to be dependent on a nonspecific adsorption of the reductase to Agarose 0.5 m such that the enzyme eluted with an apparent molecular weight of 20 000 or less. However, all batches of Agarose 0.5 m did not adsorp the reductase equally. Heller and Shrewsbury have previously shown that the reductase behaves in an anomalous manner on Agarose 0.5 m [ 41. The reasons for large variations in specific activity of ‘purified’ enzyme have not been established. However, a number of explanations for these differences can be proposed: (a) the solubilized enzyme may not have been assayed under optimal conditions. Indeed the optimal assay conditions for the reductase are different for the solubilized and microsomal enzyme [ 131; (b) the enzyme may be purified in mixtures of active and inactive forms. Nordstrom et al. [25] have recently reported that the microsomal enzyme is a mixture of active and inactive enzymes and that these two forms are interconvertable; (c) the reductase may have been copurified with another protein. The molecular weight of the partially purified reductase was found to be approximately 200 000 and the enzyme appears to be a tetramer with subunits

133

of molecular weight approximately 52 000. Previous reports have given the molecular weight of the subunit as 47 000 [7], 65 000 [8] and 120 000 [4]. Definitive proof for the preparation of a homogenous protein is often difficult to establish. However, the reported variations in the size of the reductase subunit could indicate that all investigators are not working with the same protein. The finding by Berde et al. [26] that enzyme activity did not corn&rate with the protein band after electrophoresis of enzyme purified either by the method of Heller and Shrewsbury [4] or Tormanen et al. [6] may also indicate non-homogeneity. Enzyme purified by the latter method was subsequently shown [ 71 to give two bands after electrophoresis and the enzyme activity was reported to comigrate with the major band. However, the specific activity of the enzyme in these latter studies [7] was only 12% of the specific activity reported here. Recently, the reductase obtained from rat [6,7] and chicken [9] liver, has been shown to bind to blue dextran-Sepharose. However, large affinity columns of approximately 120-500 ml were used [6,7,9] and the enzyme was eluted with NADPH [ 71. Wilson [27] has reported that the Ki values for the inhibition of a number of enzymes were lower for Cibacron Blue F3GA than for blue dextran and proposed that these differences may have been due to the steric constraints of the dextran. Our observation that large amounts of the reductase (25 000 units; specific activity, 1300 units/mg protein) were bound to small (4.0 ml) Blue Sepharose affinity columns is consistent with this proposal. Furthermore, the enzyme was bound to Blue Sepharose via the binding sites for both substrates. In this report, using purified enzyme obtained from rats fed a normal diet, the K, obtained for (S)-hydroxymethylglutaryl CoA (2.0 MM) was similar to the values reported for the microsomal [ 28,291 and purified enzyme [ 31. The K, for NADPH (65 PM) was similar to that reported by Kawachi and Rudney [3] although the specific activity was approximately 1800 fold greater in the present report. The K, for NADPH was significantly less than the value of 0.32 mM recently reported by Srikantaiah et al. 171. Comparison of the specific activities of purified enzyme obtained from animals fed a normal diet and one supplemented with cholestyramine (11 300 and 19 600, respectively, Table III), suggest that cholestyramine feeding results in a catalytically more active enzyme, the activation being approximately 80%. This was confirmed by immunotitration of solubilized enzyme obtained from rats fed 0% or 5% cholestyramine (Table IV; Fig. 5). The immunotitration data are consistent with a 40% activation of the enzyme after cholestyramine feeding. Hence, we conclude that feeding a diet containing 5% cholesyramine for four days results in an approximate 60% activation of the reductase. Cholestyramine feeding results in a 4-10 fold increase in reductase specific activity in the microsomal fraction (Table IV). Since cholestyramine feeding results in enzyme activation of approximately 60%, a 2.5-6.25 fold increase in the number of molecules of the reductase would account for the 4-10 fold increase in the microsomal specific activity. This conclusion is based on the premise that the antibody does not differentiate between the normal and activated forms of the enzyme. The conclusion that cholestyramine feeding results in enzyme activation is

134

supported by earlier studies by Higgins et al. [ 81. However, recently Kleinsek et al. [30] have reported that the enzyme from normal and cholestyramine-fed rats have similar antisera end points. The reason for this discrepancy is not known. However, data presented here from both immunotitrations and from enzyme purification indicate that cholestyramine feeding results in both enzyme activation and increased amounts of enzyme protein. The identification of the physiological intermediate of cholesterol metabolism that regulates hydroxymethylglutaryl CoA reductase activity has been elusive. However, we have demonstrated that the inactivated enzyme which is formed after mevalonolactone administration is recognized by the antibody to the reductase and results in a change in the equivalence point (Table IV). Using the antibody it may now be possible to isolate and identify the regulatory intermediate if the putative inhibitor remains bound to the antigen-antibody complex. The concentration of the reductase in the liver is extremely low. Based on the specific activities of the purified enzyme reported here and given that the microsomal content of liver is approximately 20 mg of protein/g tissue, it can be calculated that each g of liver obtained at the middle of the dark period contains approximately 3 pg or 6 pug of the reductase for normal and cholestyramine-fed rats, respectively. Presumably, the enzyme concentration is significantly lower in animals killed at the time of basal reductase activity. Such low protein concentrations emphasize the importance of developing monospecific antibody against the reductase so that the regulation of this enzyme can be understood at the molecular level. Acknowledgments We express thanks to Drs. T. Parker and E. Stellwagen for helpful discussions and to Ms. Sherri Bell for assistance in the preparation of the manuscript. These studies were supported by United States Public Health Service Research Grants Nos. HL 19063,20807 and 22474 and from Grant No. 522 from the American Heart Association, Greater Los Angeles Affiliate and from the Edna and George Castera Fund at UCLA. P.A.E. is an Established Investigator of the American Heart Association. A.M.F. is the recipient of Research Career Development Award HL 00426 from the United States Public Health Service. References 1

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2

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K.W.,

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3

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