Biomedical subjects
Publications and source records attributed to V W Rodwell.
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Rat liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyzes, in addition to its normal biosynthetic or forward reaction (HMG-CoA + 2 NADPH + 2H+----mevalonate + 2 NAD+ + CoASH), the reverse reaction (mevalonate + CoASH + 2 NADP+----HMG-CoA + 2 NADPH + 2H+) and two "half-reactions" that involve the presumed intermediate mevaldate (mevaldate + CoASH + NADP+----HMG-CoA + NADPH + H+ and mevaldate + NADPH + H+----mevalonate + NADP+). These reactions were studied using both enzyme solubilized by the traditional freeze-thaw method and enzyme solubilized with a nonionic detergent in the presence of inhibitors of proteolysis. All four reactions were inhibited by mevinolin, a known inhibitor of the forward (biosynthetic) reaction catalyzed by HMG-CoA reductase. When the enzyme was inactivated by ATP and a cytosolic, ADP-dependent HMG-CoA reductase kinase, the rates of both the forward reaction and the half-reactions decreased to comparable extents. Although coenzyme A is not a stoichiometric participant in the second half-reaction (mevaldate + NADPH + H+----mevalonate + NADP+), it was required as an activator of this reaction. This observation implies that coenzyme A may remain bound to the enzyme throughout the normal catalytic cycle of HMG-CoA reductase.
Pseudomonas sp. M grown on mevalonate as the sole source of carbon has 200- to 800-fold induced levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The enzyme, which was purified to a homogeneous state in 54% yield (final specific activity, 60.5 mumol of NAD+ reduced per min per mg of protein), converted R-mevalonate (Km = 0.15 mM) to S-HMG-CoA. Activity was sensitive to sulfhydryl modifying reagents. The apparent molecular weight of the holoenzyme was 178,000 and that of the subunit 43,000. The enzyme thus appears to be a tetramer. Comparison of a 23-residue amino-terminal sequence with the cDNA-derived sequence of Chinese hamster ovary cell HMG-CoA reductase showed little homology and antibody raised against the Pseudomonas enzyme did not appear to cross-react with rat liver HMG-CoA reductase. Addition of mevalonate to cells growing on glucose was followed by a rapid and biphasic induction of HMG-CoA reductase activity. During phase I, mevalonate or its catabolites may accumulate in intact cells of Pseudomonas sp. M and acetoacetate, a competitive inhibitor of HMG-CoA reductase (Ki = 3.2 mM), may feedback inhibit the enzyme under these conditions.
Rat hepatocytes were used to demonstrate rapid, transient effects on the modulation state (defined as the fraction of the enzyme present in the catalytically active form) of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, E.C. 1.1.1.34). Insulin elevated, while glucagon, cAMP or cGMP lowered HMG-CoA reductase modulation state within 10 to 15 min. These changes were accompanied by a parallel change in sterol synthesis. Total HMG-CoA reductase activity was not altered. Rapid modulation of HMG-CoA reductase activity therefore constitutes a viable in vivo control mechanism. By contrast to the hormones and second messengers, mevalonolactone lowered both HMG-CoA reductase modulation state and total reductase quantity.
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Extensively purified rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase was used to examine the role of ADP in inactivation of HMG-CoA reductase (EC 1.1.1.34). Solubilized HMG-CoA reductase was a suitable substrate for HMG-CoA reductase kinase. At sufficiently high concentrations of solubilized HMG-CoA reductase, reductase kinase activity approached that measured using microsomal HMG-CoA reductase as substrate. Inactivation of solubilized HMG-CoA reductase by HMG-CoA reductase kinase required both MgATP and ADP. Other nucleoside diphosphates, including alpha, beta-methylene-ADP, could replace ADP. HMG-CoA reductase kinase catalyzed phosphorylation of bovine serum albumin fraction V by [gamma-32P]ATP. This process also required a nucleoside diphosphate (e.g. alpha, beta-methylene-ADP). Nucleoside diphosphates thus act on HMG-CoA reductase kinase, not on HMG-CoA reductase. For inactivation of HMG-CoA reductase, the ability of nucleoside triphosphates to replace ATP decreased in the order ATP greater than dATP greater than GTP greater than ITP, UTP. TTP and CTP did not replace ATP. Both for inactivation of HMG-CoA reductase and for phosphorylation of bovine serum albumin protein, the ability of nucleoside diphosphates to replace ADP decreased in the order ADP greater than CDP, dADP greater than UDP. GDP did not replace ADP. Nucleoside di- and triphosphates thus appear to bind to different sites on HMG-CoA reductase kinase. Nucleoside diphosphates act as allosteric activators of HMG-CoA reductase kinase. For inactivation of HMG-CoA reductase by HMG-CoA reductase kinase, Km for ATP was 140 microM and the activation constant, Ka, for ADP was 1.4 mM. The concentration of ADP required to modulate reductase kinase activity in vitro falls within the physiological range. Modulation of HMG-CoA reductase kinase activity, and hence of HMG-CoA reductase activity, by changes in intracellular ADP concentrations thus may represent a control mechanism of potential physiological significance.
Pseudomonas sp. M, isolated from soil by elective culture on R,S-mevalonate as the sole source of carbon, possessed an inducible transport system for mevalonate. This high-affinity system had a pH optimum of 7.0, a temperature optimum of 30 degrees C, a Km for R,S-mevalonate of 88 microM, and a V max of 26 nmol of mevalonate transported per min/mg of cells (dry weight). Transport was energy dependent since azide, cyanide, or m-chlorophenylhydrazone caused complete cessation of transport activity. Transport of mevalonate was highly substrate specific. Of the 16 structural analogs of mevalonate tested, only acetoacetate, mevinolin, and mevaldehyde significantly inhibited transport. Growth of cells on mevalonate induced transport activity by 40- to 65-fold over that observed in cells grown on alternate carbon sources. A biphasic pattern for cell growth, as well as for induction of mevalonate transport activity, was observed when mevalonate was added to a culture actively growing on glucose. The induction of transport activity under these conditions began within 30 min after the addition of mevalonate and reached 60% of maximal activity during phase I. A further increase in mevalonate transport activity occurred during phase II of growth. Glucose was the preferred carbon source for growth during phase I, whereas mevalonate was preferred during phase II. Only one isomer of the R,S-mevalonate mixture appeared to be utilized, since growth ceased after 45 to 50% of the total mevalonate was depleted from the medium. However, nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO2. These results suggest that mevalonate or a mevalonate catabolite may accumulate in cells of Pseudomonas sp. M during phase I and that glucose metabolism may inhibit or repress the expression of enzymes further along the mevalonate catabolic pathway.
A growing body of evidence indicates that 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34, reductase) is degraded by proteolytic enzymes during solubilization by traditional freeze-thaw techniques. We have solubilized reductase in an active, stable form with nonionic detergents [Lubrol WX or poly(oxyethylene) ether type W-1]. Solubilization proceeded in high (greater than 70%) yield in the presence of the proteolytic inhibitors leupeptin, phenylmethanesulfonyl fluoride, and ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid and was independent of prior freeze-thawing of the microsomes. We have purified detergent-solubilized reductase 40-fold in high yield by means of sucrose density gradient centrifugation and dye-ligand chromatography. Detergent-solubilized reductase is heat labile, unlike reductase solubilized by the freeze-thaw method. Detergent-solubilized reductase can be inactivated up to 90% by use of reductase kinase. This inactivation requires both adenosine 5'-triphosphate and adenosine 5'-diphosphate, as has been previously observed for both microsomal and freeze-thaw solubilized reductase. Inactivation is reversed by subsequent treatment with a phosphoprotein phosphatase.
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Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.
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For precise determination of the catalytic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), the HMG-CoA employed as substrate must be free of HMG, CoA, and other inhibitors of HMG-CoA reductase activity. The standard purification of HMG-CoA by paper chromatography gives poor resolution of HMG-CoA from CoA and may be accompanied by some decomposition of HMG-CoA. We describe a simplified procedure for synthesis and for isolation from the reaction mixture of homogeneous, high specific activity [3(-14)C]HMG-CoA free of HMG, CoA, or nonpolar contaminants. Isolation of HMG-CoA utilizes ion-exchange chromatography in a gradient of ammonium formate, which is subsequently removed by lyophilization. The methods are proposed for use in the preparation or isolation of HMG0CoA.
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We have investigated the comparative biochemistry of in vitro regulation of HMG-CoA reductase (EC 1.1.1.34) in microsomal preparations from the livers of nine vertebrates. In all instances, reductase activity was rapidly and profoundly decreased by addition of MgATP. Reductase activities were restored to near or above initial levels after removal of MgATP and incubation with a crude, low molecular weight phosphatase preparation from rat liver cytosol. Restoration of reductase activity was inhibited both by NaF and by pyrophosphate, known inhibitors of phosphoprotein phosphatase activity. Liver cytosol of species other than the rat exhibits reductase phosphatase activity. The converter enzymes that catalyze modulation of MG-CoA reductase activity (reductase kinase and reductase phosphatase) thus appear to be ubiquitous in vertebrate liver. Interconversion in vitro of active and inactive forms of reductase probably is general for vertebrate liver also. The majority of the reductase present in vertebrate liver may be present in a catalytically inactive or latent form in vivo. Under the experimental conditions used, the fraction present in the active form is, for a given species, quite constant. Species to species, from 20-45% of the reductase appears to be present in the active form.
Incubation of horse liver aldehyde dehydrogenase (aldehyde:NAD oxidoreductase, EC 1.2.1.3) with 2-hydroxyethyl disulfide formed mixed-disulfides between protein sulfhydryl groups and beta-mercaptoethanol. Reduction of aldehyde dehydrogenase activity may be associated with formation of one, or at most two, mixed-disulfides per dehydrogenase subunit. Characteristically in the case of a mixed-disulfide, inactivation was was reversed by addition of thiols. Other disulfides also inactivated aldehyde dehydrogenase. The pseudo first-order rate constants for the forward and reverse reactions (aldehyde dehydrogenase + 2-hydroxyethyl disulfide in equilibrium or formed from modified aldehyde dehydrogenase + beta-mercaptoethanol) were 0.70 and 2 liter mole-1 sec-1, respectively. The equilibrium constant was approximately 0.4. After extended incubation under conditions expected to result in complete modification of aldehyde dehydrogenase, 30% of the initial catalytic activity remained. This suggests that 2-hydroxyethyl disulfide-treated aldehyde dehydrogenase retains catalytic activity and that the sulfhydryl group modified by 2-hydroxyethyl disulfide is not essential for aldehyde dehydrogenase activity.
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Methods were developed for the assay of hydroxymethylglutaryl-CoA reductase (NADPH) activity in microsomes from rat leukocytes. The activity in freshly isolated leukocytes is low compared to rat liver but can be assayed reliably. The patterns of response of leukocyte reductase in the assay to variation in substrate concentration, protein concentration, and time mimic those of rat liver reductase. Reductase activity in leukocyte microsomes, as in liver microsomes, is depressed by dietary cholesterol and by fasting and is elevated by dietary cholestyramine. Unlike liver reductase, leukocyte reductase activity does not exhibit a detectable diurnal rhythm. We conclude that the assay of reductase in freshly isolated leukocytes holds promise as a technique for detecting the effects of various factors on cholesterol synthesis in vivo.