Clofibrate - inducible rat hepatic P 450 s IVAl and IVA 3 catalyze the a - and ( a - 1 ) - hydroxylation of fatty acids and the w - hydroxylation of prostaglandins El and F 2 a

Cytochromes P45OIVA1 and IVA3 display 72 % amino acid sequence similarity and are expressed in livers of rats treated with the hypolipidemic drug clofibrate. The catalytic activities of IVAl and IVAS were examined by cDNA-directed expression using vaccinia virus. cDNA-expressed IVAl and IVAS had relative M,s of 51,500 and 52,000, respectively, on SDSpolyacrylamide gels. Both enzymes displayed reduced, CObound absorption spectra with X, , of 452.5 nm. IVAl and IVAJ hydroxylated lauric acid at the o and 0-1 positions with equivalent w/w-1 ratios of about 12.5. IVAl had a substrate turnover of 21 min-' which was about fourfold higher than that of IVAS. The w and w-1 hydroxylation of palmitic acid was also catalyzed by these P450s with combined turnover numbers for both metabolites of 45 min-' or 18 min-' for IVAl and IVAJ, respectively. The do-1 oxidation ratio of IVAl for palmitate was 1.25 which was almost fourfold higher than that obtained for IVA3. These enzymes also catalyzed w oxidation of the physiologically important eicosanoids prostaglandins E, and Fza with turnover numbers of about one-tenth those calculated for fatty acid oxidations. No 0-1 hydroxy metabolites were produced. m These studies indicate that the P450 enzymes IVAl and IVAJ are able to catalyze the oxidations of both fatty acids and prostaglandins. -Aoyama, T., J. P. Hardwick, S. Imaoka, Y. Funae, H. V. Gelboin, and F. J. Gonzalez. Clofibrateinducible rat hepatic P450s IVAl and IVAS catalyze the oand (w-1)-hydroxylation of fatty acids and the a-hydroxylation of prostaglandins El and FZa. J. Lipid h. 1990. 31: 1477-1482. Supplementary key word w-hydroxylases Administration of the hypolipidemic drug clofibrate to rats results in the proliferation of peroxisomes and endoplasmic reticulum and the induction of a number of peroxisomal enzymes and a few specific forms of cytochrome P450s (1-4). This induction process is mediated, in part, through a transcriptional mechanism (5, 6) . A P450, designated P 4 5 0 ~ ~ , or IVA1' has been purified and found to catalyze the hydroxylation of lauric acid (4, 6). By use of antibody against IVA1, two proteins with relative M,s of 52,000 and 51,500 have been detected on Western immunoblots (6 , 7), the latter of which corresponds to IVA1. A cDNA to IVAl mRNA was sequenced and found to encode a P450 with the expected lauric acid hydroxylase activity (6) . The nature of the second 52,000 dalton immunorelated protein has remained elusive. Recently, two additional genes in the IVA subfamily, designated IVA2 and IVA3, were identified (8, 9). By use of oligonucleotides specific for each mRNA, it was determined that all three IVA genes are induced in liver by clofibrate (9). The IVA2 mRNA is constitutively expressed in kidney while the IVAl and IVAJ mRNAs were present at low levels and inducible by clofibrate in this organ. Based on similarities in amino terminal protein sequence, it appeared that a P450 purified from livers of diabetic rats, designated P450 DM-2, that catalyzes lauric acid hydroxylation, is equivalent to IVAS (10). In the present study we used vaccinia virus to carry out expression of the IVAJ cDNA. The expressed protein was found to react with antibody against P450 K-5, to have a relative M, of 52,000 on SDS-polyacrylamide gels, and to catalyze the wand (w-1)-hydroxylation of lauric acid and palmitic acid and the w-hydroxylation of prostaglandins E, and F 2 a . Abbreviations: PBS, phosphate-buffered saline; HPLC, high perfor'The nomenclature used in this report is that described by Nebert et mance liquid chromatography.