cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity.
We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.
Yokotani
N
N
Department of Chemistry, Faculty of Science, Tohoku University, Aoba Sendai, Japan.
Kusunose
E
E
Sogawa
K
K
Kawashima
H
H
Kinosaki
M
M
Kusunose
M
M
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification*; D005227:Fatty Acids / Q000378:metabolism*; D006900:Hydroxylation; D007527:Isoenzymes / Q000235:genetics*; D007668:Kidney / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits
D005227:Fatty Acids; D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D003577:Cytochrome P-450 Enzyme System