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).

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Antibodies have been prepared to rat hepatic cytochrome P‐450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P‐450 in vitro and in situ in various components of the endoplasmic reticulum. A preparation of heavy rough endoplasmic reticulum translocates proteins newly bio‐synthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P‐450. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P‐450 can be found in the intracisternal content of heavy rough, light rough and smooth endoplasmic reticulum and also in a solubilized Golgi preparation. We suggest that at least part of the newly biosynthesized cytochrome P‐450 is translocated into the intracisternal space of the rough endoplasmic reticulum and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.

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