Solubilization and partial characterization of rat liver squalene epoxidase.
The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003579:Cytochrome Reductases / Q000378:metabolism; D003840:Deoxycholic Acid; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic; D005182:Flavin-Adenine Dinucleotide; D006358:Hot Temperature; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility; D013185:Squalene
D004988:Ethers, Cyclic; D003840:Deoxycholic Acid; D005182:Flavin-Adenine Dinucleotide; D011092:Polyethylene Glycols; D013185:Squalene; D010105:Oxygenases; D003579:Cytochrome Reductases