Isolation and purification of a rat liver 3-hydroxy-3-methylglutaryl-coenzyme reductase activating protein (RAP).
A protein with an estimated subunit mass of 19 kDa was isolated and purified from perfused rat liver cytosol. This protein activates hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (NADPH) (EC 1.1.1.34), the rate-limiting enzyme in the cholesterol biosynthetic pathway. The activation process by this HMG-CoA reductase activating protein (RAP) is time-dependent and requires NADPH. Maximal activity of HMG-CoA reductase induced by RAP is comparable to that obtained in the presence of thiols, such as GSH, and can exceed 100-fold the activity obtained when thiols are omitted. Purified RAP lacks ability to reduce 5,5'-dithiobis-(2-nitrobenzoic acid). RAP was purified to homogeneity utilizing DEAE- and phenyl-Sepharose CL-4B column chromatography. The purified RAP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shows multiple interconvertible aggregational forms on native polyacrylamide gel electrophoresis. A monospecific antibody against RAP was prepared by immunization of hens and extracted from either their egg yolks or serum. The catalytic activity of RAP might be responsible for the physiological activation of HMG-CoA reductase and regulation of its activity.
Dotan
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Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Shechter
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D000818:Animals; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification*; D051381:Rats
D000911:Antibodies, Monoclonal; D009249:NADP; D004228:Dithionitrobenzoic Acid; C054373:HMG-coenzyme A reductase activator protein, rat; D010088:Oxidoreductases; D006903:Hydroxymethylglutaryl CoA Reductases; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent