Effects of clofibrate on the main regulatory enzymes of cholesterogenesis

The in vivo effect of clofibrate on the main regulatory enzymes of cholesterogenesis has been comparatively studied for the first time in chick liver and brain. 3-Hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase from chick liver were significantly inhibited by this hypocholesterolenic drug, while mevalonate kinase and mevalonate 5-phosphate kinase were not affected. No enzyme from chick brain was significantly inhibited by the in vivo treatment. However, both liver and brain reductase activity was inhibited in vitro by clofibrate, inhibition that was progressive with increasing concentrations (1.25-5.00 mM) of drug.     

Hypolipidemic activity of dipyridamole: effects on the main regulatory enzyme of cholesterogenesis.

The in vivo dipyridamole treatment for 16 days produced a significant decrease in chick plasma cholesterol, mainly due to the esterified form. This effect was especially patent in the VLDL + LDL fraction. Similar results were observed in triglyceride content. To our knowledge, this is the first report on this hypolipidemic effects of dipyridamole. Total and esterified cholesterol increased after the same treatment in chick liver, while brain cholesterol content was not affected. Hepatic 3-hydroxy-3- methylglutaryl-CoA reductase activity was drastically reduced, while other secondary regulatory enzymes such as mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase did not change significantly. No significant differences were found in cholesterol and lipidic phosphorus from liver microsomes, so that the effect of dipyridamole on reductase activity cannot be due to modifications in cholesterol/lipidic phosphorus molar ratio. Neither of these enzyme activities was affected in vitro by dipyridamole.     

Quantitative role of different embryonic tissues in mevalonate metabolism by sterol and nonsterol pathways. Relationship with enzyme activities of cholesterogenesis

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase activities have been determined in brain, liver, intestine and kidneys from 19-day-old chick embryo. Levels of brain reductase and decarboxylase were clearly higher than those found in the other tissues assayed. However, only small differences were observed in the activity of both kinases among the different tissues. Mevalonate metabolism by sterol and nonsterol pathways has been investigated in chick embryo at the same developmental stage. Mevalonate incorporation into total nonsaponifiable lipids was maximal in liver, followed by intestine, brain and kidneys. The shunt pathway of mevalonate not leading to sterols was negligible in both brain and liver, while a clear CO2 production was observed in intestine and kidneys. Sterols running in TLC as lanosterol and cholesterol were the major sterols formed from mevalonate by brain and kidney slices, while squalene and squalene oxide(s) were found to be mainly synthesized by liver slices. Minor differences in the percentage of different sterols were observed in chick embryo intestine. The importance of free and esterified cholesterol accumulation in the different tissues on the inhibition of cholesterogenic activity is discussed.     

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.     

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).     

Mevalonate-metabolizing enzymes in Arachis hypogaea

Summary The activities of the mevalonate metabolizing enzymes-HMG-CoA reductase, mevalonate kinase, mevalonate phosphokinase and mevalonate pyrophosphate decarboxylase -were assayed with the respective substrates in green seedlings of Arachis hypogaea. MVAPP decarboxylase is the rate-limiting step among these enzymes and is inhibited by phenolic acids. Its activity in the seedlings was found to decrease in the absence of light and on treatment with abscisic acid. These results suggest that regulation of isoprene pathway in groundnut seedlings may occur at the level of mevalonate decarboxylation.Abbreviations HMG CoA 3-hydroxy-3-methyl-glutaryl coenzyme A - MVA Mevalonate - MVAP Mevalonate-5-phosphate - MVAPP Mevalonate-5-pyrophosphate - DTT Dithiothreitol - ABA Abscisic Acid     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.     

Structural analysis of mevalonate‐3‐kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

In animals, cholesterol is made from 5‐carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)‐mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate‐3‐kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)‐mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Partial purification and properties of mevalonate kinase from neonatal chick liver

Mevalonate kinase from neonatal chick liver has been partially purified by ammonium sulphate precipitation and Sephadex G100 and DEAE-cellulose fractionation. The kinetic characteristics agreed with the sequential mechanism suggested for the enzyme and provided apparent Km values of 0.01 mM for mevalonic acid and 0.25 mM for ATP. Partially purified mevalonate kinase from neonatal chick liver showed an absolute specificity for ATP. Mn2+ was a better activator than Mg2+ at low concentrations (0.1-1.0 mM). Higher Mn2+ concentrations produced a clear inhibition of mevalonate kinase. Likewise, addition of EDTA, with or without metal ions, clearly inhibited the enzymatic reaction.     

Influence of mevalonate kinase on studies of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The nature of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase has been studied. Several observations suggest that reductase inactivator preparations from both microsomes and cytosol possess mevalonate kinase activity. (1) Reductase inactivator (reductase kinase) activity copurified with mevalonate kinase activity. (2) Inactivator activity was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, known to be potent inhibitors of mevalonate kinase. (3) Addition of an excess of mevalonate completely prevented inhibition of reductase activity. (4) Formation of phosphomevalonate fully accounted for the decreased amount of mevalonate formed in the presence of inactivator and MgATP. (5) When reductase activity was measured by NADPH oxidation, no inhibition was observed. Clearly, the presence of mevalonate kinase in reductase inactivator preparations can lead to misinterpretations concerning whether reductase activity is regulated by phosphorylation-dephosphorylation. In this paper, we present several methods and approaches which can be used to critically evaluate this possibility.     

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.     

Changes in chick liver and brain mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase during development

Phosphorylation and decarboxylation of mevalonate in chick liver and brain was investigated during early post hatching stages of development. In chick liver, both mevalonate kinase and mevalonate-5-phosphate kinase increased their activity from day 5 of age while pyrophosphate decarboxylase activity remained low during the first days after hatching, increased sharply up to day 9 of age, and remained practically unchanged thereafter. The developmental pattern obtained in brain shows a slight decrease in the phosphorylation and decarboxylation of mevalonate after the first week of postnatal development. Further studies were performed using the specific substrate of mevalonate-5-pyrophosphate decarboxylase, corroborating the results obtained using mevalonate as substrate. Changes in hepatic decarboxylase were more pronounced than those observed in mevalonate-phosphorylating enzymes, thus suggesting an important role for decarboxylase in the control of cholesterogenesis during postnatal development.     

An improved purification procedure, an alternative assay and activation of mevalonate kinase by ATP

An improved procedure for the purification of pig liver mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) is described. A high-voltage electrophoresis assay was developed for mevalonate kinase. The procedure separates mevalonate from phosphomevalonate and also from diphosphomevalonate so that it can be used to measure the subsequent enzyme, phosphomevalonate kinase (EC 2.7.4.2). The assay has allowed the reassessment of the metal ion and nucleotide specificity of the pig liver enzyme. Some of the previously reported properties reflected those of the enzymes in the coupling assay rather than mevalonate kinase itself. A series of compounds were tested as activators or inhibitors of mevalonate kinase. It was found that ATP4-, arsenate and, to a smaller extent, inorganic phosphate activated the enzyme. At fixed MgATP2- (1 mM) concentrations the activation of mevalonate kinase by free ATP4- at pH 8.0 was observed at concentrations at up to 10-fold that of MgATP2- before causing any inhibition. The presence of free ATP4- resulted in a biphasic Lineweaver-Burke plot with apparent Km values for MgATP2- being 0.14 mM and 60 microM, respectively. Fluorescence measurements were consistent with the notion that the binding of excess ATP4- to the enzyme caused a conformational change.     

Reversible inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: reductase kinase and mevalonate kinase are separate enzymes

Reductase kinase and mevalonate kinase are separated by: a) ammonium sulfate fractionation; b) chromatography on agarose-Procion Red HE3B; and c) chromatography on DEAE-Sephacel. Fractions containing only reductase kinase reversibly inactivated microsomal or homogeneous HMG-CoA reductase. Fractions containing only mevalonate kinase revealed artifactual reductase kinase activity in the absence of EDTA or mevalonic acid; however, addition of EDTA or mevalonate before reductase assay completely blocked any apparent decline in HMG-CoA reductase activity. Under these conditions no dephosphorylation (reactivation) was observed by phosphatase. The combined results demonstrate unequivocally that reductase kinase and mevalonate kinase are two different enzymes and inactivation of HMG-CoA reductase is catalyzed by ATP-Mg-dependent reductase kinase.     

Inhibition of cholesterol biosynthesis by fluorinated mevalonate analogues

The conversion of mevalonate to cholesterol in rat liver homogenates (IC50 = 0.01-1.0 mM) is inhibited by 6- (I), 6,6-di- (II), and 6,6,6-trifluoromevalonate (III), as well as 4,4-difluoromevalonate (IV). Addition of compound I, III, or IV to rat liver homogenates results in the accumulation of 5-phospho- and 5-pyrophosphomevalonate. The conversion of isopentenyl pyrophosphate to cholesterol is not inhibited by the fluorinated analogues. It thus appears likely that the decarboxylation of mevalonate 5-pyrophosphate is inhibited. Rat liver homogenates catalyze the phosphorylation of I and III. The inhibition of the decarboxylation of mevalonate 5-pyrophosphate by I and III was demonstrated directly with partially purified decarboxylase. Compound I is a remarkably effective inhibitor of the decarboxylation (Ki = 10 nM). Similar results were reported by Nave et al. [Nave, J. F., d'Orchymont, H., Ducep, J. B., Piriou F., & Jung, M. J. (1985) Biochem. J. 227, 247]. It is likely that the phosphorylated or pyrophosphorylated forms of all inhibitors tested are responsible for inhibition. We also describe a chemical method for the synthesis of mevalonate 5-pyrophosphate.     

Purification and regulation of mevalonate kinase from rat liver

Mevalonate kinase may play a key role in regulating cholesterol biosynthesis because its activity may be regulated via feedback inhibition by intermediates in the cholesterol biosynthetic pathway. To study the regulation of mevalonate kinase, the enzyme was purified to homogeneity from rat liver, and monospecific antibody against mevalonate kinase was prepared. The purified mevalonate kinase had a dimeric structure composed of identical subunits, and the Mr of the enzyme determined by gel chromatography was 86,000. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit Mr was 39,900. The pI for mevalonate kinate was 6.2. The levels of mevalonate kinase protein and enzyme activity were determined in the livers of rats treated with either cholesterol-lowering agents (cholestyramine, pravastatin, and lovastatin) or with dietary modifications. Diets containing cholestyramine alone or cholestyramine and either pravastatin or lovastatin increased mevalonate kinase activity 3-6-fold. Mevalonate kinase activity decreased approximately 50% in rats treated with diets containing either 5% cholesterol or 5% cholesterol and 0.5% cholic acid. Fasting did not significantly change mevalonate kinase activity. The amount of mevalonate kinase protein in the liver was quantitated using immunoblots, and the changes in the levels of kinase activity induced by either drug treatment or by cholesterol feeding were correlated with similar changes in the levels of mevalonate kinase protein. Therefore, under these experimental conditions, mevalonate kinase activity in the liver was regulated principally by changes in the rates of enzyme synthesis and degradation.     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.