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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Research Article|
September 15 1980
Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver
Michael B. Cooper;
Michael B. Cooper
1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K.
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John A. Craft;
John A. Craft
1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K.
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Margaret R. Estall;
Margaret R. Estall
1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K.
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Brian R. Rabin
Brian R. Rabin
1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1980 London: The Biochemical Society
1980
Biochem J (1980) 190 (3): 737–746.
Citation
Michael B. Cooper, John A. Craft, Margaret R. Estall, Brian R. Rabin; Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. Biochem J 15 September 1980; 190 (3): 737–746. doi: https://doi.org/10.1042/bj1900737
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