文章基本信息

标题：Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system.
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作者：T Aoyama ; K Nagata ; Y Yamazoe 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1990
卷号：87
期号：14
页码：5425-5429
DOI：10.1073/pnas.87.14.5425
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A cDNA containing the full coding region of human cytochrome b5 was inserted into a vaccinia virus cDNA expression vector. Infection of human thymidine kinase-minus (TK-) 143 cells in culture with this recombinant virus resulted in production of 0.3 nmol of cytochrome b5 per mg of cell lysate protein. The expressed cytochrome had a reduced difference spectrum with a Soret peak at 424 nm, typical of pure cytochrome b5. TK- 143 cells have little detectable endogenous cytochrome b5, cytochrome P-450 (P450), and NADPH-P450 oxidoreductase. To test whether cytochrome b5 potentiated mixed-function monooxygenation in situ, these cells were coinfected with three recombinant vaccinia viruses individually carrying cDNAs encoding cytochrome b5, NADPH-P450 oxidoreductase, and P450 form IIB1. These triple-virus-infected cells were compared to cells infected with the P450IIB1 and NADPH-P450 oxidoreductase recombinant viruses with respect to P450IIB1-catalyzed monooxygenase activities. Cytochrome b5 specifically augmented the deethylation of p-nitrophenetole in microsomal membrane fractions of infected cells or when substrate was incubated directly with cells in situ. No significant increases were seen with P450IIB1-catalyzed testosterone, 7-ethoxycoumarin, or 7-pentoxyresorufin oxidations. These data demonstrate that cytochrome b5 is capable of specifically augmenting monooxygenase activities in intact cells.