Solubilization of the 97-kDa native form of liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was partially purified from cholestyramine-fed rats by sequential extraction of the membrane with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and polyethylene glycol nonylphenyl ether (Triton N-101) and solubilized by incorporation of the resulting insoluble protein preparation into a detergent mixture of Triton N-101 and sodium N-lauroylsarcosinate (Sarkosyl) in the presence of high salt. The purification procedure resulted in approximately a 3-4-fold increase in specific activity compared with the microsomal fraction, and the enzyme was recovered with yields as high as 63%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blotting experiment using antiserum to the purified 53,000-dalton reductase fragment showed that the major immunoreactive polypeptide had a Mr of 97,000, that expected for the native intact form of the enzyme (Chin, D. J., Gil, G., Russell, D. W., Liscum, L., Luskey, K. L., Basu, S. K., Okayama, H., Berg, P., Goldstein, J. L., and Brown, M. S. (1984) Nature 308, 613-617). In addition, the effect of various detergents on the activity and stability of the membrane-bound and the partially purified enzyme was determined, and a method for protection of the reductase from inactivation caused by the addition of anionic detergents to the assay mixture is described.
Vermilion
J L
JL
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892.
Schroepfer
G J
GJ
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000302:isolation & purification* / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D006903:Hydroxymethylglutaryl CoA Reductases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate