Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

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.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol synthesis by cholesterol sulfate in cultured fibroblasts

Although widely distributed throughout mammalian tissues, the biological function of cholesterol sulfate remains largely unknown. In these studies we have demonstrated that cholesterol sulfate suppresses de novo sterol synthesis in cultured human fibroblasts. It was further shown in these cultured cells that cholesterol sulfate is a potent inhibitor of the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis and the site at which exogenous cholesterol suppresses endogenous cholesterol synthesis. Because cholesterol sulfate inhibited sterologenesis in steroid-sulfatase deficient fibroblasts derived from patients with recessive X-linked ichthyosis, it was inferred that cholesterol sulfate per se and not cholesterol liberated by intracellular desulfation was the inhibitor in these studies. Cholesterol sulfate may be an endogenous regulator of mammalian cholesterol biosynthesis.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Molecular and Cellular Biochemistry - Within the last few years considerable evidence has accumulated which indicates that changes in HMG-CoA reductase are due primarily, if not solely, to changes...     

Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]-mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.     

Improved assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Two improvements are described for the assay of HMG CoA reductase. These are a simple synthesis of the substrate precursor HMG-3-(14)C anhydride and a double-label ((14)C and (3)H) method for determining the amount of mevalonate-3-(14)C that is formed from the substrate.     

Evidence that changes in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity are required partly to maintain a constant rate of sterol synthesis

The effects of insulin, glucagon, pyruvate, and lactate on the rate of sterol synthesis and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity were determined in hepatocytes obtained at different times of the day from rats maintained on a controlled lighting and feeding schedule. In hepatocytes from animals killed immediately before the start of the feeding period (D0 hepatocytes), the initially low activity of HMG-CoA reductase increased during incubation while that in hepatocytes prepared 6 h later (D6 hepatocytes) remained constantly high. The rates of sterol synthesis followed similar patterns of change. In both D0 and D6 cells, insulin stimulated HMG-CoA reductase but had little or no effect on the rates of sterol synthesis. In both types of cell preparation glucagon maximally suppressed HMG-CoA reductase activity at a concentration of 10(-7) M, but there was relatively little change in the rates of sterol synthesis. Both pyruvate and lactate mitigated the glucagon-mediated inhibition of HMG-CoA reductase. Each of these lipogenic precursors alone suppressed the rate of sterol synthesis in a dose-dependent manner. These changes were more apparent in the simultaneous presence of insulin and were greater in the D0 compared to the D6 hepatocytes. In the presence of lactate or pyruvate, the activity of HMG-CoA reductase was elevated, and the increase was greater when insulin was simultaneously present. In general, changes in the rate of fatty acid synthesis were positively correlated with changes in the activity of HMG-CoA reductase. These observations suggest that the latter changes are required to compensate for variations in the availability of simple precursors for sterol synthesis.     

Atorvastatin inhibits T cell activation through 3-hydroxy-3-methylglutaryl coenzyme A reductase without decreasing cholesterol synthesis

The localization of the TCR and other signaling molecules in membrane rafts (MR) is essential for the activation of T lymphocytes. MR are stabilized by sphingolipids and cholesterol. Activation of T lymphocytes leads to the confluence of small MR and the formation of an immunological synapse that is essential for sustained activation and proliferation. In this study, we investigated the effect of statins on MR and T cell activation in superantigen-stimulated human PBMC. Atorvastatin significantly inhibited cellular activation and proliferation. The binding of cholera toxin B subunit to isolated MR and to whole cells was inhibited by low doses of statins. Statins reduce the association of critical signaling proteins such as Lck and linker of activation in T cells with MR in stimulated T cells. The expression of activation markers CD69 and CD25 was inhibited. Several statin-mediated mechanisms, such as a lower stimulation with MHC-II, an inhibition of costimulation by direct binding of statins to LFA-1, a reduced secretion of cytokines, or a depletion of cellular cholesterol pools, were excluded. Inhibition of protein prenylation had a similar effect on T cell proliferation, suggesting that a reduced protein prenylation might contribute to the statin-mediated inhibition of T cell activation. Statins induce both lower levels of low-density lipoprotein cholesterol and inhibition of T cell activation, which might contribute to an inhibition of atherosclerosis.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

Endogenous sterol synthesis is not required for regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by low density lipoprotein

It has been proposed that an endogenously synthesized oxysterol mediates the regulation of cholesterol biosynthesis by low density lipoprotein in cultured mammalian cells. Studies in this report demonstrate that under conditions in which squalene conversion to sterols is blocked either by inhibition of squalene cyclization or lanosterol demethylation, or both, low density lipoprotein regulates 3-hydroxy-3-methylglutaryl coenzyme A reductase normally. These observations rule out the hypotheses that either an endogenously synthesized oxygenated cholesterol biosynthetic intermediate or epoxysterol is required to mediate the inhibition of this enzyme by low density lipoprotein.     

Clinical pharmacology of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

In this article, de novo cholesterol synthesis, its inhibition by HMG-CoA reductase inhibitors (statins) and clinical pharmacology aspects of the statins have been reviewed. Statins are available in both active and pro-drug forms. Their affinity to bind and subsequently to inhibit HMG-CoA reductase activity is approximately 3 orders of magnitude higher than that of natural substrate (HMG-CoA). All members of this group of lipid-lowering agents are, to a varying degree, absorbed from the gut. However, their bioavailability depends on their lipophobicity and their concomitant use with meals. The interaction between HMG-CoA reductase inhibitors and other lipid-lowering agents has been reviewed in more detail. One major side-effect of lipid-lowering combination therapy is myopathy with or without rhabdomyolysis. Combination of statins with gemfibrozil seems to increase risk of this adverse event, particularly in patients with renal impairment, more than combination with other lipid-lowering agents. Combination therapy with other agents including anticoagulants, antihypertensive, anti-inflammatory, oral hypoglycemic and antifungal agents as well as beta-blockers, H2 blockers, cyclosporine and digoxin has been also reviewed. The pleiotropic non-lipid lowering properties of statins and their effects on the quality of lipoprotein particles, the activities of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase as well as their possible synergistic effects with n-3 fatty acids, phytosterols, vitamin E and aspirin in reducing cardiovascular events warrant further investigation.     

Improved methods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A method for solubilizing HMG-CoA reductase is described that reproducibly yielded approximately 190% of the activity assayed in rat liver microsomes. Optimal solubilization occurred when microsomal membranes were frozen at a fixed concentration, thawed, homogenized in a buffer containing 50% glycerol, and incubated at 37 degrees C for 60 minutes. A rapid spectrophotometric assay of the reductase has been developed and the optimal conditions defined. Using this assay, the kinetics were determined for HMG-CoA reductase purified to a specific activity of 17,400 nmol NADPH oxidized per minute per mg protein.     

Microtubules and the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells.     

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.     

Isoflavones inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase in vitro

Isoflavones identified as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in soybean paste were assayed using the catalytic portion of Syrian hamster HMG-CoA reductase, and the kinetic values were measured using HMG-CoA and NADPH. The inhibition of HMG-CoA reductase by these inhibitors was competitive with HMG-CoA and noncompetitive with NADPH. Ki values for genistein, daidzein, and glycitein were 27.7, 49.5, and 94.7 microM, respectively.