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1.
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.  相似文献   

2.
The sigmoidal curves observed for rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase with NADPH as the varied substrate were markedly affected by feeding the animals diets containing colestipol, mevinolin and colestipol or cholesterol. Feeding of mevinolin and colestipol decreased the S0.5 for NADPH from 270 to 40 microM, while cholesterol feeding increased the value to 1.3 mM. Immuno-blotting analysis revealed that the Mr 100,000 form of HMG-CoA reductase predominated in cases where the S0.5 value was lowest, and the Mr 200,000 species was the major form where the S0.5 values were highest. Activation of HMG-CoA reductase by NADPH was not due to conversion of the Mr 200,000 form to the 100,000 form.  相似文献   

3.
A procedure is described for the assay of 3-hydroxy-3-methylglutaryl CoA-reductase (HMG-CoA reductase) in a large number of samples with minimal benchwork and within a 24-hr period. The Michaelis constants for HMG-CoA reductase were determined for microsomal enzyme from the liver of normal and cholesterol-fed rats and Morris hepatoma 5123C. The apparent Km D-HMG-CoA was ca. 3.5 microM and was not affected by assay temperature or cholesterol feeding. The apparent Km NADPH for microsomal HMG-CoA reductase was 10-15 microM and similarly was not affected by assay temperature. The Arrhenius plot parameters (activation energy and transition temperatures) were the same whether determined using the reaction velocity from fixed substrate concentrations or V from subtraction curves. This confirmed that values obtained using fixed saturating substrate concentrations are valid and not affected by a temperature-dependent alteration in the affinity of the enzyme for its substrates.  相似文献   

4.
The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.  相似文献   

5.
3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is the rate-limiting enzyme in the biosynthesis of cholesterol in mammals. Some microbial metabolites have been found to be HMG-CoA reductase inhibitors. Korean soybean paste is a unique food fermented by many microorganisms. The enzymatic method using the catalytic domain of Syrian hamster HMG-CoA reductase was employed for the screening of HMG-CoA reductase inhibitors. Soybean paste extract was fractionated by vacuum liquid chromatography. Fractions showing relatively high HMG-CoA reductase inhibition were further purified through Sephadex LH-20 column chromatography and C18 preparative HPLC, and the inhibitory compounds were identified as genistein, daidzein, and glycitein.  相似文献   

6.
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.  相似文献   

7.
C E Nakamura  R H Abeles 《Biochemistry》1985,24(6):1364-1376
The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

8.
The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.  相似文献   

9.
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.  相似文献   

10.
The lanosterol 14 alpha-methyl demethylase inhibitors miconazole and ketoconazole have been used to assess their effects upon cholesterol biosynthesis in cultured Chinese hamster ovary cells. In Chinese hamster ovary cells treated with either agent, an initial accumulation of lanosterol and dihydrolanosterol has been observed. At elevated concentrations, however, ketoconazole, but not miconazole, causes the preferential accumulation of 24,25-epoxylanosterol and squalene 2,3:22,23-dioxide. These metabolites accumulate at the expense of lanosterol, thereby demonstrating a second site of inhibition for ketoconazole in the sterol biosynthetic pathway. Both demethylase inhibitors produced a biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. The biphasic modulation is characterized by low levels of the drugs suppressing HMG-CoA reductase activity which is restored to either control or above control values at higher drug concentrations. This modulatory effect of the lanosterol demethylase inhibitors upon HMG-CoA reductase was not observed in the lanosterol 14 alpha-methyl demethylase-deficient mutant AR45. Suppression of HMG-CoA reductase activity is shown to be due to a decrease in the amount of enzyme protein consistent with a steroidal regulatory mechanism. Collectively, the results establish that lanosterol 14 alpha-methyl demethylation, but not 24,25-epoxylanosterol formation, is required to suppress HMG-CoA reductase in the manner described by lanosterol demethylase inhibitors.  相似文献   

11.
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.  相似文献   

12.
Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.  相似文献   

13.
Incubation of four purified rat liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase phosphatases (G. Gil, M. Sitges, and F. G. Hegardt, (1981) Biochim. Biophys. Acta663, 211–221) with HMG-CoA, CoA, NADPH, or citrate caused a concentration-dependent inactivation of the enzyme activities. HMG-CoA and CoA showed similar patterns of inactivation and at 0.5 mm of both compounds, the four reductase phosphatases were fully inhibited. Half-maximal inactivation was comprised between 0.02 and 0.1 mm of HMG-CoA and CoA. NADPH at concentration ranging between 5 and 10 mm produced complete inactivation of reductase phosphatases. Citrate at 5 mm produced full inactivation, and half-maximal inhibition ranged from 0.1 to 0.4 mm for the different phosphatases. The behavior of fluoride varied with respect to the four phosphatases: Low molecular forms were inactivated in a similar manner as described for other protein phosphatases. However, high molecular forms were slightly inactivated, and phosphatase IIa at 100 mm showed a level of activity similar to the control. The effect of KCl on the four reductase phosphatases could explain this behavior since at high concentrations, KCl (and NaCl) produced activation in both high and low molecular forms, this effect being more enhanced in high Mr reductase phosphatases. The insensitivity to fluoride of high Mr reductase phosphatases could explain the discrepancies in percentage of the active form of HMG-CoA reductase described previously in literature.  相似文献   

14.
Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.  相似文献   

15.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.  相似文献   

16.
The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) has been demonstrated both in homogenates and microsomes of the S3G strain of HeLa cells. It was increased 8- to 10-fold by the removal of serum from the growth medium. The presence of steroids, specifically of the glucocorticoid series, in the serum-less growth medium elicited an additional 100 to 345% increase over the serum-less control, whereas the addition of N6,O2'-dibutyryl adenosine 3':5'-monophosphate to the medium or dexamethasone to the assay mixture was without any stimulatory effect. Both inductions were blocked by cycloheximide and actinomycin D, suggesting a protein synthesis-dependent elevation of enzyme activity. Glucocorticoids were effective in the induction at concentrations ranging from 10(-6) to 10(-8) M and there was a demonstrated parallel between the magnitude of enzyme induction and glucocorticoid potency. The HMG-CoA reductase activities from steroid-induced and control cultures had identical assay characteristics (pH optima and apparent Km values for both NADPH and HMG-CoA). This induction of the rate-controlling enzyme of cholesterogenesis occurred despite the observation that glucocorticoids specifically depress the rate of acetate or water, but not mevalonate, incorporation into cholesterol.  相似文献   

17.
Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.  相似文献   

18.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is located in the endoplasmic reticulum (ER) and responds to rapid degradation which is regulated by mevalonate or sterols. T cell antigen receptor alpha chain (TCR alpha) is also known to be rapidly degraded within the ER. In both cases, the membrane domains of the proteins have a crucial role in their rapid degradation. In order to investigate protein degradation in the ER, we compared the degradation of HMG-CoA reductase and TCR alpha in the same Chinese hamster ovary cells. Among the protease inhibitors tested, N-acetyl-leucyl-leucyl-methioninal blocks the degradation of HMG-CoA reductase and also inhibits the degradation of TCR alpha. On the other hand, N-tosyl-L-phenylalanine chloromethyl ketone and N-carbobenzoxy-L-phenylalanine chloromethyl ketone inhibit the degradation of TCR alpha but have no effect on the degradation of HMG-CoA reductase. Diamide, a thiol-oxidizing agent, blocks the degradation of both HMG-CoA reductase and TCR alpha. Perturbation of cellular Ca2+ attenuates the rapid degradation of HMG-CoA reductase but does not affect the degradation of TCR alpha. Furthermore, thapsigargin, a selective ER Ca(2+)-ATPase inhibitor, and Co2+, a potent Ca2+ antagonist, increase the half-life of HMG-CoA reductase but not that of TCR alpha. Energy inhibitors diminish the rapid degradation of HMG-CoA reductase but not that of TCR alpha. These results suggest that although HMG-CoA reductase and TCR alpha appear to be degraded in the same subcellular compartment, the mechanisms responsible for degradation differ.  相似文献   

19.
A protein with an estimated subunit mass of 19 kDa was isolated and purified from perfused rat liver cytosol. This protein activates hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (NADPH) (EC 1.1.1.34), the rate-limiting enzyme in the cholesterol biosynthetic pathway. The activation process by this HMG-CoA reductase activating protein (RAP) is time-dependent and requires NADPH. Maximal activity of HMG-CoA reductase induced by RAP is comparable to that obtained in the presence of thiols, such as GSH, and can exceed 100-fold the activity obtained when thiols are omitted. Purified RAP lacks ability to reduce 5,5'-dithiobis-(2-nitrobenzoic acid). RAP was purified to homogeneity utilizing DEAE- and phenyl-Sepharose CL-4B column chromatography. The purified RAP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shows multiple interconvertible aggregational forms on native polyacrylamide gel electrophoresis. A monospecific antibody against RAP was prepared by immunization of hens and extracted from either their egg yolks or serum. The catalytic activity of RAP might be responsible for the physiological activation of HMG-CoA reductase and regulation of its activity.  相似文献   

20.
Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC50 values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.  相似文献   

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