Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.     

Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.     

Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells.

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.     

Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, is subject to rapid degradation which is regulated by mevalonate (MVA)-derived metabolic products. HMG-CoA reductase is an integral membrane protein of the endoplasmic reticulum, the largest nonmitochondrial pool of cellular Ca2+. To assess the possible role of Ca2+ in the regulated degradation of HMG-CoA reductase, we perturbed cellular Ca2+ concentration and followed the fate of HMG-CoA reductase and of HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase and the soluble bacterial enzyme beta-galactosidase. The degradation of HMGal mirrors that of HMG-CoA reductase, demonstrating that the membrane domain of HMG-CoA reductase is sufficient to confer regulated degradation (Skalnik, D.G., Narita, H., Kent, C., and Simoni, R.D. (1988) J. Biol. Chem. 263, 6836-6841; Chun, K.T., Bar-Nun, S., and Simoni, R.D. (1990) J. Biol. Chem. 265, 22004-22010). In this study we show that the MVA-dependent accelerated rates of degradation of HMG-CoA reductase and HMGal in cells maintained in Ca(2+)-free medium are 2-3-fold slower than the rate of degradation in cells grown in high (1.8-2 mM) Ca2+ concentration. This effect is reversed upon addition of Ca2+ to the medium. Furthermore, when cells maintained in high Ca2+ are treated with 1 microM ionomycin, the MVA-dependent accelerated degradation of HMG-CoA reductase and HMGal is also reduced about 2-3-fold. This inhibition is not due to a Ca(2+)-dependent uptake or incorporation of MVA into sterols, since these processes are not affected in the absence of external Ca2+. In addition, cobalt, a known antagonist of Ca(2+)-dependent cellular functions, totally abolishes (IC50 = 520 microM in the presence of 1.8 mM extracellular Ca2+) the MVA-accelerated degradation of HMGal. These results suggest that Ca2+ plays a major role in the regulated degradation of HMG-CoA reductase.     

Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the "linker" segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.     

The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum

A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, beta-galactosidase of Escherichia coli, has been previously constructed. This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its beta-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this report, we demonstrate that the loss of beta-galactosidase activity results from the accelerated degradation of the HMGal protein. Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection. Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold. Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism. This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein. In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity. Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum.     

Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.     

Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.     

Regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in permeabilized cells.

We have studied the regulated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase within the endoplasmic reticulum in cells permeabilized with digitonin. Using Chinese hamster ovary cells transfected with a plasmid encoding HMGal, a chimeric protein containing the membrane domain of HMG-CoA reductase coupled to beta-galactosidase, we have demonstrated mevalonate and sterol-stimulated loss of beta-galactosidase activity. In pulse-chase experiments we have demonstrated mevalonate-stimulated degradation of both HMGal and HMG-CoA reductase. The rate of mevalonate-stimulated degradation observed in permeabilized cells tends to be slightly slower than that observed in intact cells treated with mevalonate and is dependent upon incubation of cells with mevalonate prior to permeabilization. The degradation process measured in this report extends a previous report of HMG-CoA reductase degradation in digitonin-permeabilized cells (Leonard, D. A., and Chen, H. W. (1987) J. Biol. Chem. 262, 7914-7919) by mimicking key physiological features of the in vivo process, including: stimulation by regulatory molecules, specifically mevalonate and sterols; inhibition by cycloheximide; and inhibition by an inhibitor of neutral cysteine proteases.     

The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor.

The ubiquitin-dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature-sensitive defect in ubiquitin conjugation (CHO-ts20), as well as into wild-type cells (CHO-E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO-E36 and in CHO-ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH-dependent fashion. However, at the non-permissive temperature in CHO-ts20 cells, neither GH-dependent uptake nor degradation of the GHR was observed, while in CHO-E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO-E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand-induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH-induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.     

Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.     

Inhibition of hemoglobin degradation in Plasmodium falciparum by chloroquine and ammonium chloride

The effect of chloroquine (CQ) and NH4Cl on hemoglobin degradation within Plasmodium falciparum was studied by SDS gel electrophoresis. CQ inhibited hemoglobin digestion such that accumulation of hemoglobin in the parasites occurred. Quantitative analysis indicated that the content of hemoglobin in isolated CQ-treated parasites was increased from 2.39 +/- 0.47 micrograms hemoglobin (Hb)/mg protein to 8.34 +/- 0.77 micrograms Hb/mg protein (P less than 0.001) within 45 min and further to 18.7 +/- 1.23 micrograms Hb/mg protein for 2 hr compared with the untreated parasites. These results suggest that the inhibition of hemoglobin degradation in malarial parasites might be the primary target of CQ antimalarial action. The CQ-like effect on the hemoglobin digestion in P. falciparum was observed with another lysosomotropic weak base, NH4Cl, suggesting that the CQ effect on hemoglobin degradation and its antimalarial action, as well as the effect of NH4Cl, are related to their properties of lysosomotropic weak base.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.