cDNA-directed expression of rat testosterone 7 alpha-hydroxylase using the modified vaccinia virus, T7-RNA-polymerase system and evidence for 6 alpha-hydroxylation and delta 6-testosterone formation.
The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.
Aoyama
T
T
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892.
Korzekwa
K
K
Nagata
K
K
Gillette
J
J
Gelboin
H V
HV
Gonzales
F J
FJ
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004247:DNA / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006900:Hydroxylation; D015151:Immunoblotting; D007700:Kinetics; D051381:Rats; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology; D013739:Testosterone / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*
D013739:Testosterone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C022724:testosterone 7-alpha-hydroxylase, hamster; D012321:DNA-Directed RNA Polymerases