Biochem. J. (1977) 164, 501-508 Printed in Great Britain
501
A New Method for Assaying Rat Liver Microsomal 3-Hydroxy-3-methylglutarylCoenzyme A Reductase Activity and its Application in a Study of the Effect of Dietary Cholesterol on this Enzyme By YASIN A. BAQIR* and ROGER BOOTH Department ofBiochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, U.K. (Received 28 October 1976) A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected lih after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase.
Liver microsomal 3-hydroxy-3-methylglutarylCoA reductase (EC 1.1.1.34) catalyses the following reaction: 3-Hydroxy-3-methylglutaryl-CoA+ 2NADPH + 2H+ -- mevalonate + 2NADP++ CoA The rat liver enzyme shows a diurnal variation in activity (Hamprecht et al., 1969; Shapiro & Rodwell, 1971; Edwards & Gould, 1972; Gregory et al., 1972), reaching at the daily maximum a specific activity of about 1.0, and at the daily minimum of about 0.1. Specific activity is defined as units/mg of microsomal protein, where one unit of activity converts 1 nmol of 3-hydroxy-3-methylglutaryl-CoA into mevalonate in 1 min. Because only small amounts of products can conveniently be generated, the activity of this enzyme has generally been measured by incubating microsomal suspensions with radioactively labelled 3-hydroxy-3-methylglutaryl-CoA and NADPH, then isolating the labelled mevalonate formed by t.l.c. (Shapiro et al., 1969), g.l.c. (Hamprecht & Lynen, 1970) or electrophoresis (Berndt & Gaumert, 1971). Although highly sensitive, these assays are exceedingly slow because of the necessary separative step. It is not possible to make use of the oxidation of NADPH during the reductase reaction, because microsomal suspensions oxidize NADPH even when no other substrate is added (Gillette et al., 1957; Das et al., 1968). The initial rate of this 'NADPH oxidase' activity has been estimated as about 4nmol/min per mg of microsomal protein (Das et al., 1968) and there is no known way of selec* Present address: Department of Medicine, Ninewells Hospital Medical School, University of Dundee, Dundee DD2 lUD, Scotland, U.K. Vol. 164
tively inhibiting it. The possibility of assaying the reductase by measuring the formation of CoA during its reaction was therefore investigated and an enzymic assay for CoA, using 2-oxoglutarate dehydrogenase (EC 1.2.4.2), was used (Garland et al., 1965); the results are described in the present paper. The second part of this study concerns the effect of dietary cholesterol on rat liver microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity. Although this effect is well-documented, the precise mechanism whereby the activity of this enzyme is lowered is not clearly understood. Evidence suggests that the change in enzyme activity reflects both inhibition independent of any alteration in amount of enzyme protein and lowering of the amount of enzyme protein caused by a change in the rate of its biosynthesis (Higgins & Rudney, 1973), but there is doubt about how dietary cholesterol initiates these effects. Cholesterol added to microsomal preparations in vitro in a number of different experimental situations did not inhibit the reductase activity (Linn, 1967; Shapiro & Rodwell, 1971), so that no direct action by cholesterol on the enzyme has been demonstrated. More recently, studies have been made of cells in vitro. For example, the reductase in cultured fibroblasts is inhibited by low-density lipoprotein (Brown et al., 1974). Inhibition of the reductase has been demonstrated after the addition of certain lipoproteins to cultured liver cells (Breslow et al., 1975), and cholesterol or cholesterol derivatives are known to inhibit the reductase in cultured hepatoma cells (Bell et al., 1976). Work on isolated liver cells has demonstrated a rise in 3-hydroxy-3-methylglutaryl-CoA reductase activity correlated with loss of cholesterol from the
502
cells caused by incubating them in a sterol-free medium (Edwards et al., 1976). But to assess the extent to which these effects in vitro are related to the effect of dietary cholesterol in vivo, it is essential that the characteristics of this effect in vivo be fully known. In this context an experiment designed to define accurately the time course of this inhibition was therefore carried out and the results are described in the present paper. Experimental Animals Female Wistar rats, weighing 90-lOOg on arrival, were purchased from Bantin & Kingman, Grimston, Aldbrough, Yorkshire, U.K. Except for animals used to study the effect of dietary cholesterol on liver 3-hydroxy-3-methylglutaryl-CoA reductase, rats were kept for at least 2 weeks after arrival in a room artificially illuminated between 15:00h and 03:00h, and in darkness between 03:00h and 15:00h, and allowed continuous access to food and water. Unless otherwise specified, rats were killed between 09:00h and 10:00h on the day of an experiment, and the microsomal fraction was prepared from their livers as described by Tata (1969). Microsomal pellets were washed once by resuspension in homogenizing buffer (1 ml of buffer for the microsomal fraction from 1 g of liver), and recentrifugation. Microsomal protein was measured by the method of Lowry et al. (1951), with dry bovine serum albumin as standard. Reagents
3-Hydroxy-3-methylglutaryl-CoA was prepared from 3-hydroxy-3-methylglutaric acid anhydride and CoA under the conditions described by Stegink & Coon (1968), except that a 3-fold excess of the anhydride was used to minimize the amount of unchanged CoA. After acidification ofthe 3-hydroxy3-methylglutaryl-CoA solutions, excess of 3-hydroxy3-methylglutaric acid was extracted with dry peroxide-free ether. 3-Hydroxy-3-methylglutaric acid anhydride was prepared from 3-hydroxy-3-methylglutaric acid as described by Goldfarb & Pitot (1971). Before use, 3-hydroxy-3-methylglutaryl-CoA solutions were assayed by using 3-hydroxy-3-methylglutaryl-CoA reductase purified from yeast to the zinc-eluate stage of Kirtley & Rudney (1967). The reaction mixture contained 50mM-potassium phosphate (pH7.0), 10mM-2-mercaptoethanol, 0.1 mmNADPH and sufficient yeast enzyme to complete the reaction, when this was initiated by adding a sample of the unknown 3-hydroxy-3-methylglutaryl-CoA solution, within 5min. The progress of the reaction was followed spectrophotometrically at 340 nm. Alternatively, 3-hydroxy-3-methylglutaryl-CoA solu-
Y. A. BAQIR AND R. BOOTH
tions were assayed by measuring their CoA content, after alkaline hydrolysis, by using the phosphotransacetylase reaction essentially as described by Michal & Bergemeyer (1974). Both gave the same result when used on the same unknown sample. 3-Hydroxy-3-methylglutaryl-CoA prepared as described above is a racemic mixture, and only one form is a substrate for the reductases. In this paper amounts and concentrations of 3-hydroxy-3-methylglutaryl-CoA refer to the racemic mixture. Oxoglutarate dehydrogenase was prepared essentially as described by Sanadi et al. (1952). NADPH (tetrasodium salt, grade II), NADH (disodium salt, grade II), NAD+ (freeze-dried free acid, grade II), CoA (free acid, grade I), phosphotransacetylase (Clostridium kluyveri), acetyl phosphate and lactate dehydrogenase (rabbit muscle) were purchased from Boehringer Corp., Lewes, East Sussex, U.K. 2-Oxoglutaric acid was from BDH Chemicals, Poole, Dorset, U.K., and bovine serum albumin (type F), dithiothreitol and cholesterol oleate were from Sigma Chemical Co., St Louis, MO, U.S.A. Cholesterol was from Organon Laboratories, Newhouse, Lanarkshire, Scotland, U.K. and before use was purified by recrystallization as described by Leffler & McDougald (1963). Enzyme assays Oxoglutarate dehydrogenase activity was measured spectrophotometrically as described by Linn et al. (1972). Conditions for assaying microsomal 3hydroxy-3-methylglutaryl-CoA reductase activity were the subject of the present investigation, and results of experiments which led to the adoption of the following procedure as a routine are given below in the Results section. Liver microsomal fraction (microsomal protein concentration not exceeding 3 mg/ml) was incubated in a medium containing 30mM-EDTA, 70mM-NaCl, l5mM-dithiothreitol, 1.5mM-NADPH and 0.25mM-3-hydroxy-3-methylglutaryl-CoA at pH6.8 in a final volume of 0.5ml. Blank incubations contained all components except NADPH. All incubations were carried out at 37°C for 20min, then 50,ul of 2M-HCI was added to stop the reaction and destroy remaining NADPH. Precipitated material was removed by centrifugation in an MSE Minor bench centrifuge, then CoA in the supernatant was measured essentially as described by Garland et al. (1965). A portion of the supernatant was added to a fluorimeter tube containing 1 ml of a buffer containing 50mM-potassium phosphate, 0.5 mM-MgCl2, 1.25 mM-NAD+, 1.25 mM-cysteine hydrochloride and 1 mM-2-oxoglutarate, pH7.0. Sufficient 2-oxoglutarate dehydrogenase was added to complete the reaction within 1 min, and the increase in fluorescence caused by NADH formation was measured by using a Locarte fluorimeter equipped with a mercury lamp, a primary filter transmitting
1977
RAT LIVER 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE
between 340nm and 380nm and a secondary filter transmitting above 435 nm. The instrument was calibrated by using the oxidation of NADH by lactate dehydrogenase in the presence of a spectrophotometrically standardized pyruvate solution. It was generally set to give a full-scale deflexion in response to about 10#uM-NADH. At this setting the change in deflexion resulting from the 2-oxoglutarate reaction was directly proportional to the initial concentration of CoA. CoA in blanks to the reductase assays was subtracted from that in corresponding tests before calculating the activity of 3-hydroxy-3methylglutaryl-CoA reductase. One unit of activity is defined as that releasing 1 nmol of CoA in 1 min, and specific enzyme activity is expressed as units per mg of microsomal protein. Sterol analysis Samples of microsomal suspensions containing 10-20mg of protein/ml were extracted with chloroform/methanol (2:1, v/v) essentially as described by Albrink (1960). Lipids in the chloroform phase were separated by t.l.c. on silica gel G by using light petroleum (b.p. 40-60'C)/diethyl ether/acetic acid (90:10:1, by vol.) as developing solvent. After staining with iodine vapour, spots identified by means of markers as cholesterol and cholesterol ester were scraped from the plates after the iodine had sublimed away, and eluted with chloroform. Cholesterol was measured spectrophotometrically by the method of Leffler & McDougald (1963), except that the final volume in the colour test was scaled down to 2.5ml to increase sensitivity. Cholesterol ester was measured fluorimetrically as described by Solow & Freeman (1970). The final volume in the test was 1.25ml and, by using exciting light of 520nm and measuring fluorescence at 562nm, a linear response up to 4,g of cholesterol oleate was obtained. Results Assay conditions for the microsomal reductase During preliminary experiments, blank incubations were found to contain some CoA, and since the size of the blank determines the sensitivity of the enzyme assay, the source of this CoA was identified experimentally. Omitting the microsomal fraction from the incubation mixture was found to make no difference to the amount of 'blank' CoA. Omitting the incubation at 37°C for 20min lowered the amount of'blank' CoA, and blanks incubated without 3-hydroxy-3-methylglutaryl-CoA contained no measurable CoA. The conclusion drawn was that 'blank' CoA arose partly as a contaminant in the 3-hydroxy-3-methylglutaryl-CoA used as substrate, and partly by non-enzymic hydrolysis of this substrate during incubation for enzyme assay. Complete and incubated blanks generally contained about
Vol. 164
503
3 nmol of CoA, about half of which was formed by non-enzymic hydrolysis. Fig. I shows that microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity was linear up to a microsomal protein concentration of about 3.5 mg/ml, and in Fig. 2 reductase activity is shown to be linear with incubation time up to 20min. Results of experiments in which the initial concentrations of NADPH and 3-hydroxy-3-methylglutaryl-CoA were varied are summarized in Figs. 3 and 4 respectively. NADPH concentration was found to be saturating above about 1.0mM, and 3-hydroxy-3methylglutaryl-CoA above 0.2mM. To check for possible loss of CoA during the assay, the recovery of CoA added to simulated incubation mixtures was checked in an experiment described in the legend to Fig. 5, in which the results are summarized. Recovery of CoA was quantitative, so that no correction was necessary when calculating reductase activity.
Changes in activity of liver microsomal 3-hydroxy-3methylglutaryl-CoA reductase Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is affected by several external stimuli. Dietary cholesterol lowers its activity (Linn, 1967; Shapiro & Rodwell, 1971; Edwards & Gould, 1974;
.-O-j 30
C._
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25
-0
20
Cd
Cu
0'5
-o
10
,
0
*0
e'^
0
2
3
4
5
Microsomal protein concentration (mg/ml) Fig. 1. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on the concentration of microsomal protein during incubation Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the concentration of microsomal protein was varied as shown.
504
Y. A. BAQIR AND R. BOOTH
12r
o D
04
C.) Ce
t -o
r0O
B
'0 en
c) 0
I
I
/~
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20
10
0
30
40
Incubation time (min) Fig. 2. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on the incubation time Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the incubation time was varied as shown. The same result was obtained from each of five similar experiments on different microsomal preparations.
U4I >44
0
'0
0
0.1
0.2
0.3
0.4
Initial 3-hydroxy-3-methylglutaryl-CoA concentration (mM)
Fig. 4. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on initial 3-hydroxy-3-methylglutarylCoA concentration Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase was assayed as described in the text, except that the initial 3hydroxy-3-methylglutaryl-CoA concentration was varied as shown.
30
n 0 S 20 104
'0 a0d Rl0. 0
0
0.5
1.0
1.5
2.0
Initial NADPH concentration (mM) Fig. 3. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on initial NADPH concentration Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the initial NADPH concentration was varied as shown.
0
0
20 0
u
0
Gregory & Booth, 1975) and so does starvation (Regen et al., 1966; Linn, 1967; Hamprecht et al., 1969). The diurnal variation in activity is described in the introduction. As a further check on the validity of the new enzyme assay, experiments were designed to determine whether or not it could be used to demonstrate these well-known effects. Table I shows that by using the new assay, all of these effects were detected.
20
40
60
CoA added (nmol) Fig. 5. Recovery of added CoA from incubation mixtures Incubations for 3-hydroxy-3-methylglutaryl-CoA reductase activity were prepared containing all the components described in the text, except 3-hydroxy3-methylglutaryl-CoA. Instead, various quantities of CoA were added from a CoA solution previously standardized spectrophotometrically with phosphotransacetylase. The mixtures were incubated and processed for CoA assay as described in the text.
1977
RAT LIVER 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE Time course ofthe lowering of microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity by dietary cholesterol The rats used in this experiment were trained to accept one daily meal, and the experiment was conducted as described in the legend to Fig. 6. The results of this experiment show that in control rats microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity rose from 09:30h, when food was presented, throughout the experimental period of 4h. This confirms previous observations from this laboratory (Gregory et al., 1972). Even lh after feeding, the activity of this enzyme was significantly elevated (P
501
A New Method for Assaying Rat Liver Microsomal 3-Hydroxy-3-methylglutarylCoenzyme A Reductase Activity and its Application in a Study of the Effect of Dietary Cholesterol on this Enzyme By YASIN A. BAQIR* and ROGER BOOTH Department ofBiochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, U.K. (Received 28 October 1976) A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected lih after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase.
Liver microsomal 3-hydroxy-3-methylglutarylCoA reductase (EC 1.1.1.34) catalyses the following reaction: 3-Hydroxy-3-methylglutaryl-CoA+ 2NADPH + 2H+ -- mevalonate + 2NADP++ CoA The rat liver enzyme shows a diurnal variation in activity (Hamprecht et al., 1969; Shapiro & Rodwell, 1971; Edwards & Gould, 1972; Gregory et al., 1972), reaching at the daily maximum a specific activity of about 1.0, and at the daily minimum of about 0.1. Specific activity is defined as units/mg of microsomal protein, where one unit of activity converts 1 nmol of 3-hydroxy-3-methylglutaryl-CoA into mevalonate in 1 min. Because only small amounts of products can conveniently be generated, the activity of this enzyme has generally been measured by incubating microsomal suspensions with radioactively labelled 3-hydroxy-3-methylglutaryl-CoA and NADPH, then isolating the labelled mevalonate formed by t.l.c. (Shapiro et al., 1969), g.l.c. (Hamprecht & Lynen, 1970) or electrophoresis (Berndt & Gaumert, 1971). Although highly sensitive, these assays are exceedingly slow because of the necessary separative step. It is not possible to make use of the oxidation of NADPH during the reductase reaction, because microsomal suspensions oxidize NADPH even when no other substrate is added (Gillette et al., 1957; Das et al., 1968). The initial rate of this 'NADPH oxidase' activity has been estimated as about 4nmol/min per mg of microsomal protein (Das et al., 1968) and there is no known way of selec* Present address: Department of Medicine, Ninewells Hospital Medical School, University of Dundee, Dundee DD2 lUD, Scotland, U.K. Vol. 164
tively inhibiting it. The possibility of assaying the reductase by measuring the formation of CoA during its reaction was therefore investigated and an enzymic assay for CoA, using 2-oxoglutarate dehydrogenase (EC 1.2.4.2), was used (Garland et al., 1965); the results are described in the present paper. The second part of this study concerns the effect of dietary cholesterol on rat liver microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity. Although this effect is well-documented, the precise mechanism whereby the activity of this enzyme is lowered is not clearly understood. Evidence suggests that the change in enzyme activity reflects both inhibition independent of any alteration in amount of enzyme protein and lowering of the amount of enzyme protein caused by a change in the rate of its biosynthesis (Higgins & Rudney, 1973), but there is doubt about how dietary cholesterol initiates these effects. Cholesterol added to microsomal preparations in vitro in a number of different experimental situations did not inhibit the reductase activity (Linn, 1967; Shapiro & Rodwell, 1971), so that no direct action by cholesterol on the enzyme has been demonstrated. More recently, studies have been made of cells in vitro. For example, the reductase in cultured fibroblasts is inhibited by low-density lipoprotein (Brown et al., 1974). Inhibition of the reductase has been demonstrated after the addition of certain lipoproteins to cultured liver cells (Breslow et al., 1975), and cholesterol or cholesterol derivatives are known to inhibit the reductase in cultured hepatoma cells (Bell et al., 1976). Work on isolated liver cells has demonstrated a rise in 3-hydroxy-3-methylglutaryl-CoA reductase activity correlated with loss of cholesterol from the
502
cells caused by incubating them in a sterol-free medium (Edwards et al., 1976). But to assess the extent to which these effects in vitro are related to the effect of dietary cholesterol in vivo, it is essential that the characteristics of this effect in vivo be fully known. In this context an experiment designed to define accurately the time course of this inhibition was therefore carried out and the results are described in the present paper. Experimental Animals Female Wistar rats, weighing 90-lOOg on arrival, were purchased from Bantin & Kingman, Grimston, Aldbrough, Yorkshire, U.K. Except for animals used to study the effect of dietary cholesterol on liver 3-hydroxy-3-methylglutaryl-CoA reductase, rats were kept for at least 2 weeks after arrival in a room artificially illuminated between 15:00h and 03:00h, and in darkness between 03:00h and 15:00h, and allowed continuous access to food and water. Unless otherwise specified, rats were killed between 09:00h and 10:00h on the day of an experiment, and the microsomal fraction was prepared from their livers as described by Tata (1969). Microsomal pellets were washed once by resuspension in homogenizing buffer (1 ml of buffer for the microsomal fraction from 1 g of liver), and recentrifugation. Microsomal protein was measured by the method of Lowry et al. (1951), with dry bovine serum albumin as standard. Reagents
3-Hydroxy-3-methylglutaryl-CoA was prepared from 3-hydroxy-3-methylglutaric acid anhydride and CoA under the conditions described by Stegink & Coon (1968), except that a 3-fold excess of the anhydride was used to minimize the amount of unchanged CoA. After acidification ofthe 3-hydroxy3-methylglutaryl-CoA solutions, excess of 3-hydroxy3-methylglutaric acid was extracted with dry peroxide-free ether. 3-Hydroxy-3-methylglutaric acid anhydride was prepared from 3-hydroxy-3-methylglutaric acid as described by Goldfarb & Pitot (1971). Before use, 3-hydroxy-3-methylglutaryl-CoA solutions were assayed by using 3-hydroxy-3-methylglutaryl-CoA reductase purified from yeast to the zinc-eluate stage of Kirtley & Rudney (1967). The reaction mixture contained 50mM-potassium phosphate (pH7.0), 10mM-2-mercaptoethanol, 0.1 mmNADPH and sufficient yeast enzyme to complete the reaction, when this was initiated by adding a sample of the unknown 3-hydroxy-3-methylglutaryl-CoA solution, within 5min. The progress of the reaction was followed spectrophotometrically at 340 nm. Alternatively, 3-hydroxy-3-methylglutaryl-CoA solu-
Y. A. BAQIR AND R. BOOTH
tions were assayed by measuring their CoA content, after alkaline hydrolysis, by using the phosphotransacetylase reaction essentially as described by Michal & Bergemeyer (1974). Both gave the same result when used on the same unknown sample. 3-Hydroxy-3-methylglutaryl-CoA prepared as described above is a racemic mixture, and only one form is a substrate for the reductases. In this paper amounts and concentrations of 3-hydroxy-3-methylglutaryl-CoA refer to the racemic mixture. Oxoglutarate dehydrogenase was prepared essentially as described by Sanadi et al. (1952). NADPH (tetrasodium salt, grade II), NADH (disodium salt, grade II), NAD+ (freeze-dried free acid, grade II), CoA (free acid, grade I), phosphotransacetylase (Clostridium kluyveri), acetyl phosphate and lactate dehydrogenase (rabbit muscle) were purchased from Boehringer Corp., Lewes, East Sussex, U.K. 2-Oxoglutaric acid was from BDH Chemicals, Poole, Dorset, U.K., and bovine serum albumin (type F), dithiothreitol and cholesterol oleate were from Sigma Chemical Co., St Louis, MO, U.S.A. Cholesterol was from Organon Laboratories, Newhouse, Lanarkshire, Scotland, U.K. and before use was purified by recrystallization as described by Leffler & McDougald (1963). Enzyme assays Oxoglutarate dehydrogenase activity was measured spectrophotometrically as described by Linn et al. (1972). Conditions for assaying microsomal 3hydroxy-3-methylglutaryl-CoA reductase activity were the subject of the present investigation, and results of experiments which led to the adoption of the following procedure as a routine are given below in the Results section. Liver microsomal fraction (microsomal protein concentration not exceeding 3 mg/ml) was incubated in a medium containing 30mM-EDTA, 70mM-NaCl, l5mM-dithiothreitol, 1.5mM-NADPH and 0.25mM-3-hydroxy-3-methylglutaryl-CoA at pH6.8 in a final volume of 0.5ml. Blank incubations contained all components except NADPH. All incubations were carried out at 37°C for 20min, then 50,ul of 2M-HCI was added to stop the reaction and destroy remaining NADPH. Precipitated material was removed by centrifugation in an MSE Minor bench centrifuge, then CoA in the supernatant was measured essentially as described by Garland et al. (1965). A portion of the supernatant was added to a fluorimeter tube containing 1 ml of a buffer containing 50mM-potassium phosphate, 0.5 mM-MgCl2, 1.25 mM-NAD+, 1.25 mM-cysteine hydrochloride and 1 mM-2-oxoglutarate, pH7.0. Sufficient 2-oxoglutarate dehydrogenase was added to complete the reaction within 1 min, and the increase in fluorescence caused by NADH formation was measured by using a Locarte fluorimeter equipped with a mercury lamp, a primary filter transmitting
1977
RAT LIVER 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE
between 340nm and 380nm and a secondary filter transmitting above 435 nm. The instrument was calibrated by using the oxidation of NADH by lactate dehydrogenase in the presence of a spectrophotometrically standardized pyruvate solution. It was generally set to give a full-scale deflexion in response to about 10#uM-NADH. At this setting the change in deflexion resulting from the 2-oxoglutarate reaction was directly proportional to the initial concentration of CoA. CoA in blanks to the reductase assays was subtracted from that in corresponding tests before calculating the activity of 3-hydroxy-3methylglutaryl-CoA reductase. One unit of activity is defined as that releasing 1 nmol of CoA in 1 min, and specific enzyme activity is expressed as units per mg of microsomal protein. Sterol analysis Samples of microsomal suspensions containing 10-20mg of protein/ml were extracted with chloroform/methanol (2:1, v/v) essentially as described by Albrink (1960). Lipids in the chloroform phase were separated by t.l.c. on silica gel G by using light petroleum (b.p. 40-60'C)/diethyl ether/acetic acid (90:10:1, by vol.) as developing solvent. After staining with iodine vapour, spots identified by means of markers as cholesterol and cholesterol ester were scraped from the plates after the iodine had sublimed away, and eluted with chloroform. Cholesterol was measured spectrophotometrically by the method of Leffler & McDougald (1963), except that the final volume in the colour test was scaled down to 2.5ml to increase sensitivity. Cholesterol ester was measured fluorimetrically as described by Solow & Freeman (1970). The final volume in the test was 1.25ml and, by using exciting light of 520nm and measuring fluorescence at 562nm, a linear response up to 4,g of cholesterol oleate was obtained. Results Assay conditions for the microsomal reductase During preliminary experiments, blank incubations were found to contain some CoA, and since the size of the blank determines the sensitivity of the enzyme assay, the source of this CoA was identified experimentally. Omitting the microsomal fraction from the incubation mixture was found to make no difference to the amount of 'blank' CoA. Omitting the incubation at 37°C for 20min lowered the amount of'blank' CoA, and blanks incubated without 3-hydroxy-3-methylglutaryl-CoA contained no measurable CoA. The conclusion drawn was that 'blank' CoA arose partly as a contaminant in the 3-hydroxy-3-methylglutaryl-CoA used as substrate, and partly by non-enzymic hydrolysis of this substrate during incubation for enzyme assay. Complete and incubated blanks generally contained about
Vol. 164
503
3 nmol of CoA, about half of which was formed by non-enzymic hydrolysis. Fig. I shows that microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity was linear up to a microsomal protein concentration of about 3.5 mg/ml, and in Fig. 2 reductase activity is shown to be linear with incubation time up to 20min. Results of experiments in which the initial concentrations of NADPH and 3-hydroxy-3-methylglutaryl-CoA were varied are summarized in Figs. 3 and 4 respectively. NADPH concentration was found to be saturating above about 1.0mM, and 3-hydroxy-3methylglutaryl-CoA above 0.2mM. To check for possible loss of CoA during the assay, the recovery of CoA added to simulated incubation mixtures was checked in an experiment described in the legend to Fig. 5, in which the results are summarized. Recovery of CoA was quantitative, so that no correction was necessary when calculating reductase activity.
Changes in activity of liver microsomal 3-hydroxy-3methylglutaryl-CoA reductase Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is affected by several external stimuli. Dietary cholesterol lowers its activity (Linn, 1967; Shapiro & Rodwell, 1971; Edwards & Gould, 1974;
.-O-j 30
C._
CZCu u'
25
-0
20
Cd
Cu
0'5
-o
10
,
0
*0
e'^
0
2
3
4
5
Microsomal protein concentration (mg/ml) Fig. 1. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on the concentration of microsomal protein during incubation Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the concentration of microsomal protein was varied as shown.
504
Y. A. BAQIR AND R. BOOTH
12r
o D
04
C.) Ce
t -o
r0O
B
'0 en
c) 0
I
I
/~
O
20
10
0
30
40
Incubation time (min) Fig. 2. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on the incubation time Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the incubation time was varied as shown. The same result was obtained from each of five similar experiments on different microsomal preparations.
U4I >44
0
'0
0
0.1
0.2
0.3
0.4
Initial 3-hydroxy-3-methylglutaryl-CoA concentration (mM)
Fig. 4. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on initial 3-hydroxy-3-methylglutarylCoA concentration Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase was assayed as described in the text, except that the initial 3hydroxy-3-methylglutaryl-CoA concentration was varied as shown.
30
n 0 S 20 104
'0 a0d Rl0. 0
0
0.5
1.0
1.5
2.0
Initial NADPH concentration (mM) Fig. 3. Dependence of 3-hydroxy-3-methylglutaryl-CoA reductase activity on initial NADPH concentration Liver microsomal fraction was prepared and 3hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described in the text, except that the initial NADPH concentration was varied as shown.
0
0
20 0
u
0
Gregory & Booth, 1975) and so does starvation (Regen et al., 1966; Linn, 1967; Hamprecht et al., 1969). The diurnal variation in activity is described in the introduction. As a further check on the validity of the new enzyme assay, experiments were designed to determine whether or not it could be used to demonstrate these well-known effects. Table I shows that by using the new assay, all of these effects were detected.
20
40
60
CoA added (nmol) Fig. 5. Recovery of added CoA from incubation mixtures Incubations for 3-hydroxy-3-methylglutaryl-CoA reductase activity were prepared containing all the components described in the text, except 3-hydroxy3-methylglutaryl-CoA. Instead, various quantities of CoA were added from a CoA solution previously standardized spectrophotometrically with phosphotransacetylase. The mixtures were incubated and processed for CoA assay as described in the text.
1977
RAT LIVER 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE Time course ofthe lowering of microsomal 3-hydroxy3-methylglutaryl-CoA reductase activity by dietary cholesterol The rats used in this experiment were trained to accept one daily meal, and the experiment was conducted as described in the legend to Fig. 6. The results of this experiment show that in control rats microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity rose from 09:30h, when food was presented, throughout the experimental period of 4h. This confirms previous observations from this laboratory (Gregory et al., 1972). Even lh after feeding, the activity of this enzyme was significantly elevated (P
