Purification and cDNA Cloning of Human Liver CYP4A Fatty Acid ω-Hydroxylase

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Abstract
Laurate ω-hydroxylase activity of human liver microsomes was strongly inhibited by an antibody against rabbit fatty acid ω-hydroxylase P450 4A5, and Western blot analysis with this antibody showed the presence of two immunochemically related proteins with apparent molecular weights of approximately 50 and 52 kDa in all of 14 human liver specimens examined. A fatty acid ω-hydroxylase (designated P450HLω) was purified to a specific content of 15 nmol of P450/mg of protein from microsomes of a single human liver on the basis of its laurate ω-hydroxylase activity and its reactivity with the P450 4A5 antibody. This P450HLω showed an apparent molecular weight of 52 kDa on SDS-PAGE. Furthermore, a cDNA clone (designated HL24) has been isolated 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 was identical to the NH2-terminal amino acid sequence of P450HLω except for one undetermined residue. This cDNA encoded a protein of 519 amino acids with a molecular weight of 59, 347. The amino acid sequence predicted from the cDNA showed 82% identity with that of P450 4A5. Northern blot analysis showed that the mRNA hybridized to the cDNA is expressed in the human liver and kidney. The protein expressed in yeast cells using the cDNA in an expression vector, pAAH5, showed an apparent molecular weight of approximately 52 kDa, as determined by immunoblotting with the P450 4A5 antibody, and catalyzed the ω- and (ω-1)-hydroxylation of various fatty acids, thus suggesting that the cDNA HL24 corresponds to a human liver fatty acid ω-hydroxylase, P450HLω.

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