Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family.

Lauric acid w-hydroxylase (cytochrome P-450LAw) was purified from livers of rats that had been given the hypolipidemic drug clofibrate.Immunoblot analysis with anti-cytochrome P-45OLAu antibody revealed the presence of two clofibrate-induced cytochrome P-450 proteins in both liver and kidney.This antibody was used to screen a X g t l l expression library constructed from liver mRNA isolated from rats given clofibrate.The clone, pP-45OLAw, was isolated and found to contain 2062 base pairs with an open reading frame of 509 amino acids (M.58,222).The p P -4 5 0 ~~ cDNA insert was placed in the yeast expression vector pAAH5, and the resultant plasmid expressed a protein of the same size and activity as cytochrome P -4 5 0 ~~~.The mechanism of the regulation of the cytochrome P-450LAw gene by hypolipidemic agents was examined.The rate of cytochrome P-460LAw gene transcription was increased as early as 1 h after administration of clofibrate, and this increase was followed by an elevation of cytochrome P-450LAw mRNA, immunochemically detectable protein, and cytochrome P-450LAw activity.In contrast, no increase in transcriptional activity was detected after clofibrate administration for the cytochrome P-45opCN or P-450b/e genes.The cytochrome P-450LAw has several structural features in common with other cytochrome P-4508, including a conserved cysteine-containing heme-binding fifth ligand fragment and a hydrophobic amino terminus.Overall, cytochrome P-45OLAw shared less than 35% cDNA nucleotide and amino acid similarity with cytochromes P-450c, P-450d, P-450e, and P-&OpCN, indicating that it is a member of a new cytochrome P-450 gene family.Southern blot analysis indicates that this family contains two or three genes.The cytochrome P-450 microsomal multisubstrate monooxygenase system represents a superfamily (Atchison and Adesnik, 1986;Adesnik and Atchison, 1986) of enzymes that participate in the metabolism of steroids (Hall, 1986), eicosanoid, fatty acids (Kupfer, 1980), and bile acids (Hansson and Wikvall, 1980), as well as exogenous substrates such as drugs, insecticides, and chemical carcinogens (Gelboin, 1980).