Purification and characterization of two forms of fatty acid omega-hydroxylase cytochrome P-450 from rabbit kidney cortex microsomes.
We have previously reported the isolation of two forms of cytochrome P-450 (P-450) with omega-hydroxylase activities toward prostaglandin A (PGA) and fatty acids, designated as P-450ka-1 and P-450ka-2, from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)phthalate [Kusunose, E. et al. (1989) J. Biochem. 106, 194-196]. In the present work, we have purified and characterized two additional forms of rabbit kidney fatty acid omega-hydroxylase, designated as P-450kc and P-450kd. The purified P-450kc and P-450kd had specific contents of 13 and 16 nmol of P-450/mg of protein, with apparent molecular weights of 52,000 and 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Both the forms showed absorption maxima at 450 nm in the carbon monoxide-difference spectra for their reduced forms. These P-450s efficiently catalyzed the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, in a reconstituted system containing P-450, NADPH-P-450 reductase, and phosphatidylcholine. Cytochrome b5 stimulated the reactions to only a slight extent. They had no detectable activity toward PGA and several xenobiotics tested. The two P-450s showed different peptide map patterns after limited proteolysis with papain or Staphylococcus aureus V8 protease.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshimura
R
R
Toneyama Institute for Tuberculosis Research, Osaka City University Medical School.
Kusunose
E
E
Yokotani
N
N
Yamamoto
S
S
Kubota
I
I
Kusunose
M
M
D000595:Amino Acid Sequence; D000818:Animals; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D015786:Cytochromes b5 / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D006900:Hydroxylation; D007672:Kidney Cortex / Q000187:drug effects / Q000201:enzymology*; D008168:Lung / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D011817:Rabbits; D015262:Xenobiotics
D005227:Fatty Acids; D015262:Xenobiotics; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D009251:NADPH-Ferrihemoprotein Reductase