PURIFICATION AND NH2-TERMINAL AMINO-ACID-SEQUENCES OF HUMAN AND RAT-KIDNEY FATTY-ACID OMEGA-HYDROXYLASES

A cytochrome P-450 (P-450), designated P-450HK-omega, has been isolated and purified from human kidney microsomes to a specific content of 13 nmoles of P-450 / mg of protein. P-450HK-omega showed an apparent molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Absolute spectra of the oxidized form indicated that this P-450 was largely in the low-spin state and partly in the high-spin state. It catalyzed the omega-and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate, with no activity toward prostaglandin A1, benzphetamine, 7-ethoxycoumarin, or 7-ethoxyresorufin. The first 35 NH2-terminal amino acid sequence of P-450HK-omega had about 70% homology with those of rabbit kidney fatty acid omega-hydroxylases of the P-450 IVA gene subfamily, P-450ka-1, P-450ka-2, and P-450kd, except for four undertermined residues. Moreover, Western blot and immuno-inhibition studies showed that P-450HK-omega reacted with an antibody against the rabbit kidney fatty acid omega-hydroxylase. The results suggest that P-450HK-omega is a member of the same P-450 gene family (IVA subfamily) as the rabbit enzymes. In addition, the terminal sequence of P-450HK-omega also showed 54% homology with that of P-450k-2, a fatty acid omega-hydroxylase from rat kidney microsomes. To our knowledge, this is the first time that a P-450 specific for fatty acid omega-hydroxylase activity has been isolated to homogeneity from human tissues.