Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. Catalysis of the reverse reaction and two half-reactions

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.     

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mm sucrose, 50 mm KCl, 40 mm KH₂PO₄, 30 mm EDTA, and 2 mm DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,000g (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,000g (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at ?20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at 100,000g (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents, sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.     

Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and partial purification of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

This paper describes an effective method for the solubilization of microsomal HMG-CoA reductase from rat liver. Exposing the microsomes to a freeze-thaw treatment solubilized 80% of the microsomal reductase activity. Subsequently, a 25-fold purification has led to an enzyme preparation with a specific activity of 10–14 nmoles MVA per min per mg of protein and an increased stability.     

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.     

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 X g from a cell-free homogenate of four-day-old, dark-grown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2SO4 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose. The presence of detergent, which was required at all times to maintain activity, did not interfere with the chromatographic procedures used. Sucrose density centrifugation suggested an apparent molecular mass of 180 kDa with subunits of 45 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate). The enzyme was stable at 67.5 degrees C for 30 min in the presence of glycerol, dithioerythritol and detergent. Studies of enzyme stability and activation indicate that the enzyme is a hydrophobic protein with free thiol groups that are essential for full activity. The activation energy was estimated to be 92 kJ (Arrhenius plot). Antibodies raised against rat liver and yeast hydroxymethylglutaryl-CoA (HMG-CoA) reductase failed to bind or inactivate the radish enzyme. When both HMG-CoA and NADPH concentrations were varied, intersecting patterns were obtained with double-reciprocal plots. The apparent Km values determined in this way are 1.5 microM [(S)-HMG-CoA], and 27 microM (NADPH). Concentrations of NADPH greater than 150 microM caused substrate inhibition at low HMG-CoA concentrations resulting in deviations from linearity in secondary plots. Analysis of these data and the product inhibition pattern suggest a sequential mechanism for the reduction of HMG-CoA to mevalonic acid with HMG-CoA being the first substrate binding to the enzyme, followed by NADPH.     

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

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 comigrated 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-hydroxy-3-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.     

Solubilization of the 97-kDa native form of liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was partially purified from cholestyramine-fed rats by sequential extraction of the membrane with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and polyethylene glycol nonylphenyl ether (Triton N-101) and solubilized by incorporation of the resulting insoluble protein preparation into a detergent mixture of Triton N-101 and sodium N-lauroylsarcosinate (Sarkosyl) in the presence of high salt. The purification procedure resulted in approximately a 3-4-fold increase in specific activity compared with the microsomal fraction, and the enzyme was recovered with yields as high as 63%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blotting experiment using antiserum to the purified 53,000-dalton reductase fragment showed that the major immunoreactive polypeptide had a Mr of 97,000, that expected for the native intact form of the enzyme (Chin, D. J., Gil, G., Russell, D. W., Liscum, L., Luskey, K. L., Basu, S. K., Okayama, H., Berg, P., Goldstein, J. L., and Brown, M. S. (1984) Nature 308, 613-617). In addition, the effect of various detergents on the activity and stability of the membrane-bound and the partially purified enzyme was determined, and a method for protection of the reductase from inactivation caused by the addition of anionic detergents to the assay mixture is described.     

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits.     

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.     

Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.     

Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin.

Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized. The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase. In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase. Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele. The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al. (J.-O. Hoog, H. von Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43:13-21, 1986).     

Methods for Visualization of Enzymes in Polyacrylamide Gels

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C(12)B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. beta-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o-nitrophenol from o-nitrophenyl-beta-D-galactoside, and arylsulfatase from Pseudomonas C(12)B was marked in gels by liberation of p-nitrophenol from p-nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer (Enterobacter aerogenes) and a denitrifier (Pseudomonas perfectomarinus) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes, and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus. Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels (R(f) = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels (R(f) = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus, moved further in the gels (R(f) = 0.64) than either of the enzymes from E. aerogenes.     

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.     

Solubilization and characterization of a platelet membrane ADP-binding protein.

Previous studies have shown that platelet membranes bind radiolabeled ADP and have nucleoside diphosphokinase activity which transforms added ADP to ATP. In order to further characterize these reactions, the ADP-binding and nucleoside diphosphokinase activity of purified platelet membranes were solubilized by freeze-thaw injury followed by extraction with isotonic buffered saline. Up to 80% of membrane ADP-binding activity was solubilized along with 20% of the total membrane protein, a 4-fold purification. A Millipore filter binding assay was developed to detect the soluble binding protein using [3H]ADP as radioligand. Binding of [3H]ADP was rapid, reversible, saturable, and was destroyed by heat, trypsin digestion, and 1 mM N-ethylmaleimide. By Scatchard analysis, there was a single class of binding sites with a Kd of 3.8 x 10(-7) M. Unlabeled nucleotides competed with [3H]ADP with the following potency series: ATP = ADP greater than AMP greater than adenosine. The solubilized nucleoside diphosphokinase activity could be separated from ADP-binding activity by ultracentrifugation on 5 to 20% sucrose density gradients containing 0.6 M KCl suggesting that the activities reside on separate molecules. Hydrodynamic parameters were calculated for the binding protein by gel filtration and ultracentrifugation. The s20,w was 4.1, Stoke's radius 35 x 10(-8)cm, axial ratio (f/fo) 1.09, and the Mr = 61,000. The studies suggest that this platelet ADP-binding protein may act as the receptor for initiating ADP-induced aggregation and release.     

The effect of gonadotropins on the mitochondrial adenylate cyclase of rat testis

The formation of adenosine 3′:5′-cyclic monophosphate from ATP by testicular mitochondria of immature and mature rats was increased to the same extent by addition of either human chorionic gonadotropin or luteinizing hormone. Follicle stimulating hormone was found to be more active in stimulating adenylate cyclase activity in testicular mitochondria of immature rats. The stimulatory effect of gonadotropins were not suppressed by Ca⁺⁺ complexing agent ethylene-glycol-bis-(β-amino-ethyl ether) N,N′-tetra-acetic acid. The detergent Lubrol PX, solubilized 75–80% of the mitochondrial adenylate cyclase. The solubilized enzyme was activated by sodium fluoride but not by gonadotropins. The present results indicate a direct effect of gonadotropins on the adenylate cyclase attached to mitochondrial membranes.     

A simple procedure for the solubilization of NADH-cytochrome b5 reductase

NADH-cytochrome b₅ reductase has been solubilized by extraction of rabbit liver microsomes with 1 potassium phosphate buffer (pH 7.4), and has been purified to comparable purity with the Triton X-100-solubilized enzyme. Gel electrophoresis indicated an apparent molecular weight of 33,000 for both phosphate buffer-extracted and Triton X-100-solubilized enzymes. Phosphate buffer extraction provides a simple mild procedure for the extraction of NADH-cytochrome b₅ reductase that avoids detergents or proteolytic agents.     

Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates

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.     

Synthesis, properties and reactions of 3 beta-benzoyloxy-7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-ene.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (I) with gaseous HCl in chloroform at -40 degrees C gave, in 87% yield, 3 beta-benzoyloxy-7 alpha,15 beta-dichloro-5 alpha cholest-8(14)-ene (III). Reduction of the latter compound with lithium aluminum hydride in ether at room temperature for 20 min gave, in 86% yield, 7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-en-3 beta-ol (IV). The latter compound was fully characterized and assignments of the individual carbon peaks in the 13C nuclear magnetic resonance spectra of this sterol have been completed. Reduction of III with excess lithium aluminum hydride in refluxing ether for 4 days gave, in 74% yield, 5 alpha-cholesta-7,14-dien-3 beta-ol (VI). Reduction of the dichloro-steryl benzoate III with lithium triethylborohydride in tetrahydrofuran gave, in 88% yield, 5 alpha-cholest-8(14)-en-3 beta-ol (VII). A similar reduction using lithium triethylborodeuteride led to the formation of [7 beta, 15 xi-2 H2]-VIIa. Treatment of III with concentrated HCl in a mixture of chloroform and methanol gave, in 79% yield, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one (II) which was characterized as such and as the corresponding free sterol.     

Localization of the cyclic adenosine 3' : 5' -monophosphate phosphodiesterase activator protein in rat heart.

A cyclic adenosine 3′ : 5′ — monophosphate phosphodiesterase activator protein has been partially purified from rat heart by a procedure involving ammonium sulfate fractionation and affinity column chromatography with cyclic AMP phosphodiesterase bound to Sepharose 4B. Freezing and thawing of the rat heart was essential for solubilization of the activator protein in the crude homogenate. Activator activity was localized on sarcoplasmic reticulum isolated from fresh heart which could be solubilized with a low yield that resulted in a labile product. Maximal activation of cyclic AMP phosphodiesterase with excess protein activator was 100%.