HMG-CoA reductase kinase: measurement of activity by methods that preclude interference by inhibitors of HMG-CoA reductase activity or by mevalonate kinase.
Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.
D055372:AMP-Activated Protein Kinases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008722:Methods; D009097:Multienzyme Complexes / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011919:Rats, Inbred Strains
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D009097:Multienzyme Complexes; D000244:Adenosine Diphosphate; D004492:Edetic Acid; D010770:Phosphotransferases; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases