Simultaneous expression of ferredoxin, ferredoxin reductase and P450 in COS7 cells.
cDNA fragments encoding mouse ferredoxin and ferredoxin reductase were simultaneously introduced into COS7 cells by using an expression vector, pUC-SR alpha plasmid. When using the mitochondrial fraction prepared from the transfected cells, cytochrome-c reductase activity was detected. This activity was highest when 7.5 micrograms of the ferredoxin expression plasmid (pSR alpha F) and 2.5 micrograms of the ferredoxin reductase expression plasmid (pSR alpha FR) were transfected into COS7 cells. In this system, NADPH could be replaced by NADH as a cofactor for the reduction of cytochrome-c although the cytochrome-c reductase was more dependent on NADPH than NADH at a low concentration. When CYP24 expression plasmid was transfected into COS7 cells along with both pSR alpha F and pSR alpha FR, the transfected cells revealed a 3-fold higher 25-hydroxyvitamin D3-24-hydroxylase activity than COS7 cells transfected with CYP24 expression plasmid.
Itoh
S
S
Division of Cellular Physiology, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Iemura
O
O
Yoshimura
T
T
Tsujikawa
K
K
Yamada
E
E
Nonaka
Y
Y
Okamoto
M
M
Mimura
T
T
Kohama
Y
Y
D000818:Animals; D019556:COS Cells; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000235:genetics*; D005288:Ferredoxins / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013250:Steroid Hydroxylases / Q000235:genetics; D014162:Transfection; D065668:Vitamin D3 24-Hydroxylase
D018076:DNA, Complementary; D005288:Ferredoxins; D009243:NAD; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D005287:Ferredoxin-NADP Reductase; D009245:NADH Dehydrogenase