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.     

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.     

Developmental pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the rat

The activity, protein concentration and catalytic efficiency of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was determined in rats aged 1 to 199 days. Microsomal enzyme total activity peaked on day 24, during weaning, and again on day 63, during the onset of puberty. Increased enzyme activity during weaning resulted primarily from an increase in the catalytic efficiency of the enzyme with a slight reduction in enzyme protein content. The rise in enzyme activity during the onset of puberty, however, was primarily the result of an increase in enzyme protein concentration. Thus, the activity of reductase in mammalian livers reflects, at different stages in development, the modulating influence of both the total number of reductase molecules and the catalytic efficiency of the enzyme.     

Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase

Sequence analysis has revealed two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of the Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised catalytic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of bound mevaldehyde, an inference supported by mutagenesis of lysine 267 to aminoethylcysteine. For this mechanism to be general, Class I HMG-CoA reductases ought also to possess an active site lysine. Three lysines are conserved among all Class I HMG-CoA reductases. The three conserved lysines of Syrian hamster HMG-CoA reductase were mutated to alanine. All three mutant enzymes had reduced but detectable activity. Of the three conserved lysines, sequence alignments implicate lysine 734 of the hamster enzyme as the most likely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did not reflect an altered structure. Substrate recognition was essentially normal, and both circular dichroism spectroscopy and analytical ultracentrifugation implied a native structure. Enzyme K734A also formed an active heterodimer when coexpressed with inactive mutant enzyme D766N. We infer that a lysine is indeed essential for catalysis by the Class I HMG-CoA reductases and that the revised mechanism for catalysis is general for all HMG-CoA reductases.     

Cholesterol is not synthesized in membranes bearing 3-hydroxy-3-methylglutaryl coenzyme A reductase

We have shown previously that newly synthesized lanosterol and cholesterol in homogenates of cultured human fibroblasts do not have the same equilibrium buoyant density as the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in the smooth endoplasmic reticulum (SER) (Lange, Y., and Steck, T. L. (1985) J. Biol. Chem. 260, 15592-15597). This finding suggested two alternative and novel hypotheses: (a) that lanosterol and cholesterol might be transported rapidly from the SER to other internal membranes or (b) that synthesis of the sterols is not associated with the SER, or at least not with that portion of this organelle bearing HMG-CoA reductase. We therefore compared the subcellular distribution of HMG-CoA reductase with that of enzymes which convert lanosterol to cholesterol. The two activities studied were the consumption of exogenous [3H]lanosterol and the conversion of exogenous radiolanosterol to radiocholesterol. Differential centrifugation, rate zonal centrifugation, and equilibrium sucrose gradient centrifugation of rat liver homogenates all showed that these enzyme activities did not comigrate with HMG-CoA reductase. The subcellular distribution of newly synthesized sterols also was examined in cultured human fibroblasts. Cells were incubated with radioactive acetate to label endogenous sterols biosynthetically, homogenized, and spun to equilibrium on sucrose gradients. The buoyant density profiles of radioactive cholesterol and lanosterol both had a peak at 1.12 g/cm3. Digitonin treatment shifted both sterols to higher densities, strong evidence that they resided in cholesterol-rich membranes. Pretreatment of intact cells with cholesterol oxidase, which selectively oxidizes plasma membrane cholesterol, abolished the digitonin shift of lanosterol but not of intracellular cholesterol. These findings provide support for the hypothesis that newly synthesized cholesterol and lanosterol are not in the same membrane.     

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.     

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.     

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.     

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

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.     

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.     

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.