PURIFICATION AND CDNA CLONING OF HUMAN LIVER CYP4A FATTY-ACID OMEGA-HYDROXYLASE

Laurate omega-hydroxylase activity of human liver microsomes was stron gly inhibited by an antibody against rabbit fatty acid omega-hydroxyla se P450 4A5, and Western blot analysis with this antibody showed the p resence of two immunochemically related proteins with apparent molecul ar weights of approximately 50 and 52 kDa in all of 14 human liver spe cimens examined. A fatty acid omega-hydroxylase (designated P450HL ome ga) was purified to a specific content of 15 nmol of P450/mg of protei n from microsomes of a single human liver on the basis of its laurate omega-hydroxylase activity and its reactivity with the P450 4A5 antibo dy. This P450HL omega showed an apparent molecular weight of 52 kDa on SDS-PAGE. Furthermore, a cDNA clone (designated HL24) has been isolat ed from a human liver cDNA library by using the cDNA for P450 4A5 as a probe. The sequence of residues 5 through 25 deduced from cDNA HL24 w as identical to the NH2-terminal amino acid sequence of P450HL omega e xcept for one undetermined residue. This cDNA encoded a protein of 519 amino acids with a molecular weight of 59,347. The amino acid sequenc e predicted from the cDNA showed 82% identity with that of P450 4A5. N orthern blot analysis showed that the mRNA hybridized to the cDNA is e xpressed in the human liver and kidney. The protein expressed in yeast cells using the cDNA in an expression vector, pAAH5, showed an appare nt molecular weight of approximately 52 kDa, as determined by immunobl otting with the P450 4A5 antibody, and catalyzed the omega- and (omega -1)-hydroxylation of various fatty acids, thus suggesting that the cDN A HL24 corresponds to a human liver fatty acid omega-hydroxylase, P450 HL omega.