Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation.

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.

• Publication year

  1985

• Venue

  Journal of Biological Chemistry

• Publication date

  1985-08-25

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(17)39243-8PMID 4019513
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Immunoprecipitation of [35S]methionine-labeled hepatocytes indicated a 98 kDa target size for loss of monomer subunits, supporting a noncovalently linked dimeric enzyme.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• The minimal functional size of 3-hydroxy-3-methylglutaryl-CoA reductase was unchanged by cholestyramine feeding or by 25-hydroxycholesterol plus mevalonate treatment in isolated hepatocytes.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• Radiation inactivation of isolated hepatic microsomes and microsomes from irradiated primary hepatocytes gave a single-exponential loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity corresponding to a 200 kDa functional molecular mass.

  Confidence 0.98

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

• 25-hydroxycholesterol

  sterol

  An oxysterol added to cultured hepatocytes as one of the experimental treatment conditions.

  Aliases: 25-OHC

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• [35s]methionine labeling

  labeling method

  Metabolic incorporation of radioactive methionine into hepatocyte proteins before irradiation.

  Aliases: 35S-methionine labeling

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• 3-hydroxy-3-methylglutaryl-coa reductase

  enzyme

  The rat liver enzyme measured in this experiment as the protein whose functional size was being determined.

  Aliases: HMG-CoA reductase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• cholestyramine

  drug

  A bile-acid sequestrant added to the rat diet as one of the experimental treatment conditions.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• hepatic microsomes

  cell fraction

  Microsomal membrane fractions prepared from rat liver and used as the enzyme source for activity measurements.

  Aliases: liver microsomes

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• immunoprecipitation

  assay

  An antibody-based method used here to isolate the radiolabeled reductase protein from hepatocyte extracts.

  Aliases: IP

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• mevalonate

  metabolite

  A cholesterol-pathway intermediate added with 25-hydroxycholesterol during the hepatocyte treatment condition.

  Aliases: mevalonic acid

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• noncovalently linked dimer

  oligomeric state

  A two-subunit protein assembly held together by noncovalent interactions rather than covalent bonds.

  Aliases: noncovalent dimer

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• primary hepatocytes

  cell type

  Freshly isolated rat liver cells maintained in culture and used for irradiation and enzyme recovery experiments.

  Aliases: isolated hepatocytes, rat hepatocytes

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• radiation inactivation

  method

  A sizing approach that estimates functional molecular mass from activity loss after high-energy irradiation.

  Aliases: radiation target size analysis

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

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