Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver

A procedure for the purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP⁺ oxidoreductase (CoA-acylating); EC 1.1.1.34] from rat liver microsomes has been developed. The enzyme preparations obtained by this procedure have specific activities of 16 to 23 μmol of mevalonate formed per minute per milligram of protein. These enzyme preparations were judged to be homogeneous on the basis of comigration of enzyme activity and protein on polyacrylamide gels.     

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.     

Some properties of purified 3-hydroxy-3-methylglutaryl coenzyme A reductase phosphatases from rat liver

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 M_r reductase phosphatases. The insensitivity to fluoride of high M_r reductase phosphatases could explain the discrepancies in percentage of the active form of HMG-CoA reductase described previously in literature.     

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations

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.     

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.     

Sulfhydryl/disulfide forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase

Using radiation inactivation and immunoblotting techniques, evidence for functionally active forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase with molecular weights of about 100,000 and 200,000 was obtained. In liver microsomes isolated from rats fed both mevinolin and colestipol, the Mr 100,000 form was the predominant species, whereas in microsomes from animals fed only colestipol, the Mr 200,000 species was the major form. This Mr 200,000 form could be converted to the Mr 100,000 form by addition of dithiothreitol or beta-mercaptoethanol. Although both forms appear to possess catalytic activity, the Mr 200,000 species displays sigmoidal kinetics with respect to the concentration of NADPH, whereas the Mr 100,000 form exhibits typical hyperbolic kinetics.     

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mm sucrose, 50 mm KCl, 40 mm KH₂PO₄, 30 mm EDTA, and 2 mm DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,000g (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,000g (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at ?20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at 100,000g (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents, sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.     

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.     

Regulation of rat liver cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by methoxypolyoxyethylated cholesterol

A water-soluble derivative of cholesterol, methoxypolyoxyethylated (MPOE) cholesterol, has been synthesized and used to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme in cholesterol biosynthesis. MPOE cholesterol causes a specific, rapid and linear decline in HMG-CoA reductase in cultured rat liver cells. MPOE cholesterol is not a direct allosteric inhibitor of HMG-CoA reductase, does not appear to regulate HMG-CoA reductase through changes in membrane environment, and does not change the phosphorylation state and level of activation of rat liver cell HMG-CoA reductase. In order to confirm our data, which were consistent with a model in which MPOE cholesterol regulates the amount of HMG-CoA reductase and not its activity, we made direct measurements of reductase mRNA levels. The decline in HMG-CoA reductase in MPOE cholesterol-treated rat liver cells is preceded by the rapid disappearance of HMG-CoA reductase mRNA. As a water-soluble cholesterol derivative, MPOE cholesterol represents a useful model compound for studies on the regulation of the level of HMG-CoA reductase and its cognate mRNA.     

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...     

Structural requirements for allosteric activators of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase

Several compounds containing various structural moieties of NAD(P)(H), were examined as possible effectors of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Microsomal reductase was activated with 4.5mM GSH, assayed with subsaturating NADPH concentration and increasing amounts of the tested compounds. Under these conditions, the essential and sufficient structure required to allosterically enhance the activity of the reductase is that of 5'-AMP. When the 2' position of the nucleotide is phosphorylated, this allosteric activation is diminished.     

Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase

3-Hydroxy-3-methylglutaryl-CoA lyase has been purified to homogeneity from avian liver mitochondria. Affinity chromatography of a partially purified preparation on agarose hexane 3',5'-ADP produces enzyme of high specific activity (351 units/mg). A total purification of 1750-fold over the mitochondrial matrix fraction is achieved. The purified enzyme is stable when stored in 30% glycerol with millimolar levels of dithiothreitol. Divalent cations (e.g. Mg2+, Mn2+) and thiol-protecting agents stimulate enzyme activity under assay conditions. The enzyme binds hydroxymethylglutaryl-CoA with a Km = 8 microM. Optimal enzyme activity, measured at pH = 8.9, is 7-fold higher than activity at physiological pH. The apparent molecular weight of the native enzyme, estimated by gel filtration on Sephadex G-100, is approximately 49,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a dimer, composed of 27,000-dalton subunits. Assuming one active site per subunit, a turnover number of 158 s-1 (pH 8.2; 30 degrees C) is calculated. Antibodies have been prepared against homogeneous hydroxymethylglutaryl-CoA lyase. Ouchterlony double diffusion patterns verify the homogeneity of the preparation. Incubation of enzyme with antiserum results in virtually complete inhibition of enzyme activity.     

Reversible inactivation-reactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase of rat intestine

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the ileum of rats was inactivated by Mg2+-ATP and reversibly reactivated by cytoplasmic activator from the liver. The mevalonate kinase reaction was presumably not involved in this inactivation. Studies of nucleotide specificity for the inactivation revealed that ATP was most effective in the reaction among the nucleotides tested. In contrast to the hepatic microsomal HMG-CoA reductase, more than one-half of intestinal reductase existed in an active form. These observations indicated the presence of phosphorylation-dephosphorylation mechanism for modulation of intestinal HMG-CoA reductase.     

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.     

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.