Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity

• Published in: Molecular pharmacology Vol. 31; no. 2; pp. 152 - 158
• Main Authors: CHURCHILL, P. F, CHURCHILL, S. A, MARTIN, M. V, GUENGERICH, F. P
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.02.1987
• Subjects: Animals; Biological and medical sciences; Cloning, Molecular; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System - genetics; DNA - genetics; General pharmacology; Immunologic Techniques; Medical sciences; Microsomes, Liver - enzymology; Mixed Function Oxygenases - genetics; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; Polymorphism, Genetic; Rats; Rats, Inbred Strains - genetics; RNA, Messenger - genetics; Sex Factors; Hydroxylation; Rat; Cytochrome P450; Liver; Rodentia; Sex; Strain; Vertebrata; Mammalia; Messenger RNA; Enzymatic activity; Complementary DNA; Animal; Drug-metabolizing enzyme
• ISSN: 0026-895X; 1521-0111

A rat liver cDNA library was prepared using the expression vector bacteriophage lambda gt11 and plaques were screened using polyclonal antibodies raised to purified rat liver cytochrome P-450UT-H, the major enzyme involved in debrisoquine 4-hydroxylation, bufuralol 1'-hydroxylation, and sparteine delta 5-oxidation. A clone was selected which contained a 1.3-kb insert. The Escherichia coli beta-galactosidase fusion protein had a molecular weight greater than that of native beta-galactosidase (and reacted with anti-P-450UT-H after electrophoresis) and was also shown to compete with microsomal P-450UT-H for anti-P-450UT-H, partially relieving catalytic inhibition by anti-P-450UT-H in rat liver microsomes. Hybrid selection experiments with the cloned cDNA also support the view that the insert is related to P-450UT-H. mRNA electrophoresis/hybridization experiments indicated that the 1.3-kb cDNA probe recognized primarily only a single size class of mRNA (2.0 kb) in rat liver. mRNA blotting and in vitro translation/immunoprecipitation experiments both indicated that levels of P-450UT-H mRNA are similar in male and female Sprague-Dawley rats. Dark Agouti strain rats of both sexes contained significantly less P-450UT-H mRNA than did Sprague-Dawley rats and the females had approximately one-half the level of the males. These results are consonant with sex and strain differences in measured levels of P-450UT-H and bufuralol 1'-hydroxylase and sparteine delta 5-oxidase activities. Analysis of genomic DNA indicated that several DNA restriction fragments hybridized to this partial length cDNA; no differences were found between the rat strains and sexes. The results suggest that the basis for the variation in P-450UT-H and its activities among rat strains and sexes is at the level of mRNA concentrations.