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

• Published in: The Journal of biological chemistry 1987-01, Vol.262 (2), p.801-810
• Main Authors: Hardwick, J P, Song, B J, Huberman, E, Gonzalez, F J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; cDNA; clofibrate; Clofibrate - pharmacology; cloning; cytochrome P-450; Cytochrome P-450 CYP4A; DNA Restriction Enzymes; Fundamental and applied biological sciences. Psychology; gene expression; gene family; gene regulation; Genes; Genes. Genome; Kinetics; liver; Male; Microsomes, Liver - drug effects; Microsomes, Liver - enzymology; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Molecular and cellular biology; Molecular genetics; nucleotide sequence; Rats; Rats, Inbred Strains; transformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)75857-8

Lauric acid omega-hydroxylase (cytochrome P-450LA omega) was purified from livers of rats that had been given the hypolipidemic drug clofibrate. Immunoblot analysis with anti-cytochrome P-450LA omega antibody revealed the presence of two clofibrate-induced cytochrome P-450 proteins in both liver and kidney. This antibody was used to screen a lambda gt11 expression library constructed from liver mRNA isolated from rats given clofibrate. The clone, pP-450LA omega, was isolated and found to contain 2062 base pairs with an open reading frame of 509 amino acids (Mr 58,222). The pP-450LA omega 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-450LA omega. The mechanism of the regulation of the cytochrome P-450LA omega gene by hypolipidemic agents was examined. The rate of cytochrome P-450LA omega gene transcription was increased as early as 1 h after administration of clofibrate, and this increase was followed by an elevation of cytochrome P-450LA omega mRNA, immunochemically detectable protein, and cytochrome P-450LA omega activity. In contrast, no increase in transcriptional activity was detected after clofibrate administration for the cytochrome P-450PCN or P-450b/e genes. The cytochrome P-450LA omega has several structural features in common with other cytochrome P-450s, including a conserved cysteine-containing heme-binding fifth ligand fragment and a hydrophobic amino terminus. Overall, cytochrome P-450LA omega shared less than 35% cDNA nucleotide and amino acid similarity with cytochromes P-450c, P-450d, P-450e, and P-450PCN, 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.