Purification of 2,3-oxidosqualene cyclase from rat liver.
A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.
Moore
W R
WR
Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Schatzman
G L
GL
D000818:Animals; D002845:Chromatography / Q000379:methods; D003902:Detergents; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats
D003902:Detergents; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase