Evaluation of monooxygenase induction as a means of enhancing the yield of the rat liver cytochrome P-450 isoenzyme with high affinity for cimetidine.
The binding of cimetidine to liver microsomes prepared from untreated rats and rats pretreated with phenobarbitone or 3-methylcholanthrene has been investigated by difference spectroscopy and equilibrium partition methods. In M/15 phosphate buffer, pH 7.9, microsomes from each group of rats yielded markedly biphasic spectral binding curves, which have been interpreted in terms of two independent classes of cytochrome P-450 site with widely differing binding affinities for the drug. Support for such interpretation was provided by the finding that the spectral binding curve for a purified sample of the principal cytochrome P-450 isoenzyme from liver microsomes of phenobarbitone-pretreated rats could be described adequately by a single rectangular hyperbolic relationship, the spectral dissociation constant being indistinguishable experimentally from that for the weaker class of cytochrome P-450 binding site in the corresponding microsomes. The spectral dissociation constants were 2 microM and 80 microM for microsomes from untreated rats; 44 microM and 540 microM for those from phenobarbitone-pretreated rats; and 34 microM and 540 microM for microsomes from rats pretreated with 3-methylcholanthrene. On this basis, both classes of P-450 site in the microsomes from rats subjected to either pretreatment exhibited lower affinity for cimetidine than their counterparts in microsomes from untreated rats. Equilibrium partition studies of the higher-affinity class of microsomal binding site for cimetidine showed that the twofold increase in the cytochrome P-450 content of microsomes effected by 3-methylcholanthrene pretreatment was more than offset by a diminished proportion of the total cimetidine-binding capacity present as the higher-affinity, pharmacologically significant, receptor (18%, cf. 48% in control microsomes); and that phenobarbitone pretreatment resulted in replacement of the high-affinity receptor by one with a threefold weaker cimetidine-binding affinity. Thus the use of these monooxyginase inducers to enhance the cytochrome P-450 content of liver microsomes would seem to offer little potential in the isolation of the isoenzyme with high affinity for cimetidine.
D000818:Animals; D002927:Cimetidine / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010105:Oxygenases / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D002927:Cimetidine; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases