Yokotani, Noboru, Kusunose, Emi, Sogawa, Kazuhiro, Kawashima, Hidenori, Kinosaki, Masahiko, Kusunose, Masamichi, and Fujii-Kuriyama, Yoshiaki
We have recently purified three distinct forms of fatty acid ω-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 eDNA 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, 21665-21669]. The present paper describes cloning, sequencing and expression of a eDNA for the third fatty acid, ω-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P450kd 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 eDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled laurie acid was added to the culture medium of COS-7 cells transfected with the eDNA, significant amounts of radioactive dodecanedioic acid, together with ω- and (ω-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the ω- and (ω-1)-hydroxylation of laurie acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P450kd 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. [ABSTRACT FROM AUTHOR]