Open AccesscDNA cloning and expression of the mRNA for cytochrome P ‐450 kd which shows a fatty acid ω‐hydroxylating activity
Author(s) -
YOKOTANI Noboru,
KUSUNOSE Emi,
SOGAWA Kazuhiro,
KAWASHIMA Hidenori,
KINOSAKI Masahiko,
KUSUNOSE Masamichi,
FUJIIKURIYAMA Yoshiaki
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb15846.x
Subject(s) - complementary dna , microbiology and biotechnology , biology , cdna library , lauric acid , amino acid , messenger rna , biochemistry , peptide sequence , hydroxylation , cytochrome , cytochrome p450 , fatty acid , gene , enzyme
We have recently purified three distinct forms of fatty acid ω‐hydroxylase cytochrome P ‐450 ( P ‐450), designated P ‐450 ka‐1 , P ‐450 ka‐2 and P ‐450 kd , from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P ‐450 ka‐1 and P ‐450 ka‐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 cDNA for the third fatty acid, ω‐hydroxylase, P ‐450 kd , from a rabbit kidney cDNA library. The cDNA for P ‐450 kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P ‐450 ka‐1 . Its deduced NH 2 ‐terminal sequence of amino acids 5–24 is in complete agreement with the NH 2 ‐terminal sequence of P ‐450 kd . The identity of the cDNA was further confirmed by its expression in COS‐7 cells. When 14 C‐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 ω‐ and (ω‐1)‐hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the ω‐ and (ω‐1)‐hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P ‐450 kd gave a single band at the approximately 2.6‐kb position. The mRNA for P ‐450 kd 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 ‐450 kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.