Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver.
1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH-cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH-cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH-cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH-cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D004790:Enzyme Induction / Q000187:drug effects; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D013481:Superoxides / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D013481:Superoxides; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D010634:Phenobarbital