Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system.
Rabbit antibodies raised against the hydrophilic part of microsomal NADPH-cytochrome P450 oxidoreductase (denoted fpT) demonstrated a marked ability to inhibit NADPH-sterol Delta7-reductase activity. In addition, trypsin and proteinase K treatment of microsomes removed almost all microsomal electron transfer constituents from the microsomes, but the Delta7-reductase activity could be reconstituted by adding detergent-solubilized NADPH-cytochrome P450 oxidoreductase (denoted OR). Furthermore, after solubilization from microsomes, the Delta7-reductase activity could be reconstituted with OR in a DEAE-cellulose column chromatography eluate fraction, which contained little OR activity. In the microsomal system, carbon monoxide, ketoconazole, and miconazole, specific inhibitors of cytochrome P450, had no effect on Delta7-reductase activity. These results provide the first evidence of an essential requirement of OR, which is distinct from cytochrome P450, in the NADPH-sterol Delta7-reductase system. EDTA, o-phenanthroline and KCN markedly lowered Delta7-reductase activity in a dose-dependent manner. Among metal ions tested, only ferric ion restored the reductase activity in the EDTA-treated microsomes. These results sugguest that NADPH-sterol Delta7-reductase is membrane-bound iron-dependent protein embedded in the microsomal lipid bilayer.
Nishino
H
H
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, 060-8638, Japan.
Ishibashi
T
T
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D000906:Antibodies; D002413:Cations, Divalent; D002614:Chelating Agents; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019995:7-dehydrocholesterol reductase; D009251:NADPH-Ferrihemoprotein Reductase