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

PF Churchill, SA Churchill, MV Martin and FP Guengerich

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.

Volume 31, Issue 2, pp. 152-158, 02/01/1987
Copyright © 1987 by American Society for Pharmacology and Experimental Therapeutics