Solubilization and purification of β-hydroxy-β-methylglutaryl coenzyme A reductase from rat liver

Kawachi, Takashi; Rudney, Harry

doi:10.1021/bi00810a008

Cited by 103 publications

(10 citation statements)

References 17 publications

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“…Usually the incubation at 37°C of membranes in the presence of Brij W-1, glycerol and dithioerythritol resulted in an increase of measurable HMGR activity, possibly due to the reduction of essential thiol groups [22] and by stabilizing hydrophobic domains of the enzyme. Since the release of the HMGR from rat liver microsomes by the usual freeze-thawing procedure requires the activity of lysosomal membrane-bound proteases [28], we checked the role of proteolysis in the freezethaw steps of the solubilization procedure.…”

Section: Solubilizution and Purification Of Hmgr Activitymentioning

confidence: 99%

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

1986

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3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 x g from a cell-free homogenate of four-day-old, darkgrown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2S04 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose. The presence of detergent, which was required at all times to maintain activity, did not interfere with the chromatographic procedures used. Sucrose density centrifugation suggested an apparent molecular mass of 180 kDa with subunits of 45 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate). The enzyme was stable at 67.5T for 30 min in the presence of glycerol, dithioerythritol and detergent. Studies of enzyme stability and activation indicate that the enzyme is a hydrophobic protein with free thiol groups that are essential for full activity. The activation energy was estimated to be 92 kJ (Arrhenius plot). Antibodies raised against rat liver and yeast hydroxymethylglutarylCoA (HMG-CoA) reductase failed to bind or inactivate the radish enzyme. When both HMG-CoA and NADPH concentrations were varied, intersecting patterns were obtained with double-reciprocal plots. The apparent K,,, values determined in this way are 1.5 pM [(S)-HMG-CoA], and 27 pM (NADPH). Concentrations of NADPH greater than 150 pM caused substrate inhibition at low HMG-CoA concentrations resulting in deviations from linearity in secondary plots. Analysis of these data and the product inhibition pattern suggest a sequential mechanism for the reduction of HMG-CoA to mevalonic acid with HMG-CoA being the first substrate binding to the enzyme, followed by NADPH. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR)regulates the synthesis of mevalonic acid, the specific precursor of the myriad of isopentenoid compounds functional in plant biochemical processes. In mammalian cells this enzyme is generally regarded to be the most important ratecontrolling enzyme for cholesterogenesis. Because of the close relation between increased serum cholesterol levels and atherosclerosis in man and the possible involvement of HMGR in the regulation of the cell cycle, this enzyme has generated a great deal of interest [I -61. Comparably little information is available on the properties and regulation of plant HMGR. In recent work [7-91 it has been shown that, in radish seedlings, HMGR activity is Correspondence to T.

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Section: Solubilizution and Purification Of Hmgr Activitymentioning

confidence: 99%

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

1986

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“…A number of procedures have been reported for the solubilization and partial purification of HMG-CoA reductase from the microsomal membrane (4)(5)(6)(7)(8). In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9)(10)(11).…”

mentioning

confidence: 99%

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

Kleinsek¹,

Ranganathan²,

Porter³

1977

Proc. Natl. Acad. Sci. U.S.A.

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A procedure for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341 solubilized from rat liver microsomes is reported. This enzyme has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This represents a 4100 fold purification over the activity in microsomes, and a specific activity that is approximately 20-fold greater than the highest previously reported value. The enzyme is judged to be homogeneous on the basis of sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, polyacrylamide disc gel electrophoresis, and immunoanalysis. Data are also presented that indicate the separation of enzymatically active and inactive species of 3-hydroxy-3-methylglutaryl-coenzyme A reductase on affinity chromatography on a coenzyme A column.3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34] catalyzes the reduction of HMG-CoA to mevalonic acid, the rate-limiting step of cholesterol biosynthesis in liver (1-3). Therefore, a number of researchers have focused their attention on the regulation of this enzyme. However, to study the modulation of HMG-CoA reductase under various physiological states a method is required to quantitate the amount of enzyme present. This can be achieved by either direct isolation of the enzyme by a reproducible purification procedure or by immunoprecipitation using monospecific antiserum to the enzyme.A number of procedures have been reported for the solubilization and partial purification of HMG-CoA reductase from the microsomal membrane (4-8). In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9-11). However, the enzyme activities of these preparations were very low (10-516 nmol of mevalonate formed per min/mg of protein).In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein. In this paper we report the purification of HMG-CoA reductase from rat liver by a combination of standard protein fractionation steps and coenzyme A affinity chromatography. This preparation also has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This value is approximately 20-fold greater than the best value previously reported.As a part of this study we also show that enzymatically active and inactive species of HMG-CoA reductase are separated by affinity chromatography. This separation suggests the possibility that cholesterol synthesis may be regulated in vvo by the interconversion of these species.MATERIALS AND METHODS Materials. Chemicals were obtained from the following sources: 3-hydroxy-3-methyl[3-'4C]glutaric acid, New England Nuclear; coenzyme A, thioester-linked agarose-hexane-coenzyme A, and dithiothrei...

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“…The affected site in all these cases appears to be HMGCoA reductase in the endoplasmic reticulum from which microsomes are derived. These compounds do not show the inhibition of the enzyme activity when added to microsomal preparations in assay mixtures, an effect similar to that of cholesterol [13]. In the case of cholesterol, it is also known from the work of Gil et al [14] that a portion of the trans-membrane domain (4-6 helices) is necessary for cholesterol-dependent degradation of HMGCoA reductase.…”

Section: Introductionmentioning

confidence: 90%

“…Clofibrate [7], cholesterol [13], deithylhexylphthatate [10] and silatrane [12] may be cited as examples. Experiments in our laboratory have indicated that these agents fail to inhibit the activity even when microsomal membrane organization is sought to be perturbed with detergents.…”

Section: Many Hypocholesterolemic Agents Which Inhibitmentioning

confidence: 99%

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

et al. 1992

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On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58 kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50 kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58 kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50 kDa protein) to the membrane, present in the 58 kDa-protein appears to be involved in mediating the inhibition by silatrane.

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Solubilization and purification of β-hydroxy-β-methylglutaryl coenzyme A reductase from rat liver

Cited by 103 publications

References 17 publications

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

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

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

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