RT Journal Article
SR Electronic
T1 Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 152
OP 158
VO 31
IS 2
A1 P F Churchill
A1 S A Churchill
A1 M V Martin
A1 F P Guengerich
YR 1987
UL http://molpharm.aspetjournals.org/content/31/2/152.abstract
AB 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.