Cytochrome P‐450 isozyme/isozyme functional interactions and NADPH‐cytochrome P‐450 reductase concentrations as factors in microsomal metabolism of warfarin

Abstract

The basis for our previous observations [Kaminsky, L. S., Guengerich, F. P., Dannan, G. A. & Aust, S. D. (1983) Arch. Biochem. Biophys. 225, 398 -4041 that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P-450 (P-450) isozymes reconstituted with NADPH -cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency 2 P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism ofthat the reductase and P-450 isozymes may be microsomal preparations.warfarin by the various P-450 isozymes. The results suggest located differently relative to one another in the various The first resolution of the hepatic monooxygenase system into solubilized fractions comprising cytochrome P-450, NADPH -cytochrome P-450 reductase, and a heat-stable component was reported in 1968 [l]. Reconstitution of the solubilized and purified components of hepatic monooxygenases into systems which catalyse the metabolism of a wide variety of compounds was subsequently extensively investigated [2, 31. The interaction of highly purified P-450 [4 -61 and NADPH -P-450 reductase [7 -91 from PB-induced rats in a reconstituted system is reversible, governed by mass action, and leads to a 1 : 1 complex (dissociation constant approximately 0.05 pM) which is essential for catalysis [lo, 113. The association between the cytochrome and reductase is slow relative to the rate of electron transfer [12] although this conclusion may be an artifact of the methods used. Some studies with reconstituted systems have been interpreted to support the hypothesis of a fluid lipid matrix for the P-450 isozymes and reductase in the microsomal membrane, where the enzymes have rapid lateral mobility [13]. Other studies Correspondence to L. S . Kaminsky Abbreviutions. P-450, microsomal cytochrome P-450; PB, phenobarbital; BNF, 2-naphthoflavone; PCN, pregnenolone-16a-carbonitrile; 3-MC, 3-methylcholanthrene; H...

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

“…Substrate-dependent activation or inhibition of one or both isoforms has been observed. These P450-P450 interactions have also been observed in triple-expression systems and microsomes isolated from rabbits after administration of a selective inducer (Kaminsky and Guengerich, 1985;Tan et al, 1997;Li et al, 1999;Cawley et al, 2001).…”

“…Substrate-dependent activation or inhibition of one or both isoforms has been observed. These P450-P450 interactions have also been observed in triple-expression systems and microsomes isolated from rabbits after administration of a selective inducer (Kaminsky and Guengerich, 1985;Tan et al, 1997;Li et al, 1999;Cawley et al, 2001).Direct physical evidence of interactions between P450s, defined as the formation of heteromers, has been more challenging to establish mainly because of their lipophilic nature and the tendency to aggregate. CYP1A1-CYP3A2 and CYP2E1-CYP2C2 complexes were identified using bimolecular fluorescence complementation spectroscopy and chemical cross-linking followed by immunoprecipitation, respectively (Alston et al, 1991;Ozalp et al, 2005).…”

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle

“…Several studies have shown that the metabolism of numerous substrates by a given P450 enzyme is altered in the presence of other P450s (21,25,(42)(43)(44). The specific interaction with mixed reconstituted systems containing both CYP1A2 and FIGURE 4.…”

Interactions among Cytochromes P450 in Microsomal Membranes

“…Competition of different P450 species for a limited amount of CPR, which has been observed by numerous investigators (1)(2)(3)(4)(5)(6), constitutes an important element in the inter-P450 cross-talk. However, the integrative connections in microsomal monooxygenase go far beyond that and involve functional effects of physical interactions of multiple P450 molecules that result in oligomerization (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”