Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

Influence of a naturally occurring competing enzymic activity on studies of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

An enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for D-hydroxymethylglutaryl CoA has been found in isolated rat liver microsomes and in microsomal extracts. The presence of this activity in enzyme preparations causes a decrease in the rate of mevalonate formation leading to an underestimation of reductase activity and an overestimation of the apparent Km of the reductase. The product formed by this competing enzymic activity behaves similarly to, but not identically with, mevalonolactone when chromatographed on Bio-Rad AG 1-x8 formate, which is used in many reductase assay procedures to separate mevalonolactone from hydroxymethylglutaryl CoA. Removal of this competing enzymic activity from reductase preparations can be accomplished by gel filtration using Bio-Gel A 1.5m, by washing the microsomes or by incubating the microsomal extract at 37 degrees C. Using enzyme preparations free of this competing enzymic activity, the apparent Km values of the reductase for D-hydroxymethylglutaryl CoA and NADPH were found to be 1.3 and 26 micronM respectively.     

Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.

A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

HMG CoA reductase of intestinal mucosa and liver of the rat

Methods were developed for the determination of HMG CoA (3-hydroxy-3-methylglutaryl CoA) reductase activity in subcellular fractions of intestinal mucosa and liver of Wistar strain rats. In the liver, reductase activity was located exclusively in the microsomal fraction. In the intestinal mucosa, activity was found in both mitochondrial and microsomal fractions of crypt cells but not of villi. The microsomal HMG CoA reductases of liver and intestinal mucosa had similar kinetic characteristics and pH optima. However, the activity of the hepatic enzyme differed with age and sex of the experimental animals while that of the intestinal crypt cells did not. Cholestyramine treatment enhanced the activity of the microsomal HMG CoA reductase in both liver and intestinal mucosa. Reductase activity of the intestinal crypt cells was elevated in both jejunum and ileum. The greatest stimulation, both relatively and absolutely, was observed in the distal half of the jejunum.     

Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings.

The properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the microsomal fraction of Pisum sativum seedlings have been described. The enzyme requires NADPH for activity and NADH does not support the reaction. The presence of a thiol compound such as dithiothreitol, is required for activity and a concentration of 10 mmm is optimal. The pH optimum is 6.8 and the K_m (apparent) for dl-3-hydroxy-3-methylglutaryl coenzyme A is about 100 μm.Activity of the enzyme is not affected by mevalonic acid at the concentrations tested (up to 1.0 mm). 3-Hydroxy-3-methylglutaric acid and free CoA cause substantial inhibition, whereas gibberellic acid has no effect.The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase is twice as high in etiolated seedlings as in green seedlings. In green seedlings activity is highest in the apical bud, declines sharply in semimature leaves, and there is almost no activity in mature leaves.     

In situ determination of the functional size of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase by radiation inactivation analysis

Radiation inactivation analysis of liver pieces yielded a target size of 210 kDa for hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) from rats fed a normal diet. Feeding a diet containing mevinolin and colestipol, which causes a marked increase in enzyme activity, resulted in a reduction of the target size to 120 kDa. These results are consistent with those obtained by radiation inactivation and immunoblotting analysis of isolated microsomes and suggest that the increase in HMG-CoA reductase activity caused by these dietary agents is accompanied by a change from a dimer to a monomer form of the enzyme.     

Modes of action and combination effects of polychlorinated biphenyls and gamma-hexachlorocyclohexane on the regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of polychlorinated biphenyls, gamma-hexachlorocyclohexane and the effect of a combination of these substances on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase were investigated. As known from previous investigations polychlorinated biphenyls interfere with the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat liver via enzyme-lipid interaction and at the pretranslational level. In contrast to polychlorinated biphenyls, gamma-hexachlorocyclohexane did not alter the lipid status of the microsomal membrane. Thus the location of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, and consequently the catalytic activity of the enzyme was not changed. As with polychlorinated biphenyls, gamma-hexachlorocyclohexane interacted with enzyme regulation at the pretranslational level. Northern dot hybridization experiments showed a decrease in the level of m-RNA coding for 3-hydroxy-3-methylglutaryl coenzyme A reductase. The effect of combination of gamma-hexachlorocyclohexane and polychlorinated biphenyls was not additive. The gamma-hexachlorocyclohexane effect appeared to play a more important role than that of the polychlorinated biphenyls. The results indicate that the combination effects are as important as the effects of the single compounds when making risk assessments for xenobiotics.     

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-¹⁴C] acetate, but not of [2-¹⁴C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.     

Improved methods for the study of hepatic HMG CoA reductase: one-step isolation of mevalonolactone and rapid preparation of endoplasmic reticulum.

Two new methods are described for the study of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. (1) Endoplasmic reticulum was rapidly prepared by diluting a 10,000 g supernatant with buffer containing 8 mM calcium chloride. The yield of protein and the specific activity of HMG CoA reductase in the pellet subsequently obtained by low speed centrifugation were nearly identical to those in the microsomal pellet prepared by ultracentrifugation. This technique may be particularly useful in studies of the rapid, in vitro modulation of the enzyme. (2) Mevalonolactone was extracted into benzene from the HMG CoA reductase assay mixture with an efficiency of 58%. There was less than 1% extraction of HMG CoA, acetoacetate, or beta-hydroxybutyrate. The extracted mevalonolactone was at least 98% pure as judged by thin-layer chromatography with four different solvent systems. These improved methods should significantly aid studies of the physiological importance of HMG CoA reductase.     

Interference with the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and its properties by a microsomal enzymic activity.

Rat liver microsomes and microsomal extracts contain an enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for 3-hydroxy-3-methylglutaryl coenzyme A. The presence of this activity in enzyme preparations causes errors in the determination of reductase activity and its properties. This contaminant can be removed by gel filtration using Bio-Gel A 1.5m, by washing the microsomes, or by incubating the microsomal extract at 37 °C. The K_m's of the reductase (free of this competing enzymic activity) for d-3-hydroxy-3-methylglutaryl coenzyme A and NADPH are 1.3 and 26 μm, respectively.     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.     

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).     

Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat.

In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

Stimulation of hepatic 3-hydroxy-3-methylglutaryl CoA reductase on administration of ATP

The depressed activity of hepatic 3-hydroxy-3-methylglutaryl CoA reductase in starved or cholesterol fed rats was stimulated on intraperitoneally administering small quantities of ATP.     

Hepatic cholesterol synthesis and hydroxymethylglutaryl CoA reductase activity after injection of methylazoxymethanol acetate

Cholesterol synthesis and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) in the liver of rats at various times (7, 22, 45 and 314 days) after injection with the carcinogen, methylazoxymethanol acetate (MAMA) is reported. Seven days after treatment, an increase in both cholesterol synthesis and HMG-CoA reductase activity was observed. Elevated HMG-CoA reductase activity and reduced dietary feedback was present 22 days after carcinogen. Cholesterol synthesis was normal at this time but dietary cholesterol failed to significantly reduce synthesis. Forty-five days after carcinogen both cholesterol synthesis and HMG-CoA reductase activity had returned to normal. Both parameters were normal 314 days after carcinogen. The enzyme gamma-glutamyl transferase was also elevated at 7, 22 and 314 days. These results indicate that HMG-CoA reductase activity and cholesterol synthesis exhibit different regulatory characteristics during the early stages of hepatocarcinogenesis initiated by MAMA injection.