Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system.
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.
Aoyama
T
T
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Nagata
K
K
Yamazoe
Y
Y
Kato
R
R
Matsunaga
E
E
Gelboin
H V
HV
Gonzalez
F J
FJ
D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013937:Thymidine Kinase / Q000172:deficiency; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D013937:Thymidine Kinase