Functional expression of microsomal and mitochondrial cytochrome P-450 (d and SCC) in COS-7 cells from cloned cDNA.
Using full length cDNA introduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7 cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. We have succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochrome P-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COS cells remain to be studied. These results show the applicability of the COS-7 expression system to investigations of the functions of members of the P-450 superfamily whose cDNA has been newly isolated.
Minowa
O
O
Department of Biochemistry, Japanese Foundation for Cancer Research, Tokyo, Japan.
Sogawa
K
K
Higashi
Y
Y
Fujii-Kuriyama
Y
Y
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D003080:Cold Temperature; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000235:genetics; D008861:Microsomes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010957:Plasmids; D013053:Spectrophotometry; D013347:Subcellular Fractions; D014162:Transfection
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D002786:Cholesterol Side-Chain Cleavage Enzyme; D009251:NADPH-Ferrihemoprotein Reductase