FORMATION OF LIGAND AND METABOLITE COMPLEXES AS A MEANS FOR SELECTIVE QUANTITATION OF CYTOCHROME-P450 ISOZYMES

The suitability of triacetyloleandomycin (TAO) metabolite complex formation and metyrapone binding to reduced cytochrome P450 as a means for selective isozyme quantitation has been studied. Although isozymes of both subfamilies bind metyrapone in the reduced state, selective quantitation of 2B isozymes through the metyrapone complex is possible after complex formation of P450 3A with a TAO metabolite. Thus, consecutive application of both reactions allows the spectroscopic quantitation of P450 3A and 2B isozymes. Complete conversion of P450 3A into the complex, a precondition for P450 3A quantitation, requires NADH in addition to NADPH. A precise collective quantitation of 3A + 2B isozymes as metyrapone complexes alone is not possible because the corresponding complexes possess different molar extinction coefficients, i.e 71.5 and 52 mM-1 cm-1 at 446-490 nm, respectively. The formation of the TAO complex appears to be quite specific, since it correlates well with 3A-specific enzymatic activities, i.e. TAO N-demethylation and formation of 2beta-hydroxy-, 15beta-hydroxy- and 6-dehydrotestosterone. P450 3A levels in liver microsomes of male rats either untreated or treated with TAO, dexamethasone (DEX), phenobarbital or hexachlorobenzene amount to 13%, 78%, 66%, 24% and 11% of total P450, respectively. Good correlation between these values and P450 3A-specific enzymatic activities is obtained. By the spectroscopic method, P450 2B isozymes could not be detected in microsomes of untreated rats. With TAO, DEX and hexachlorobenzene the microsomal 2B level is elevated to about 20% of total P450, i.e. to 0.8, 0.4 and 0.4 nmol P450/mg protein, respectively. 2B levels of about 60% of total P450 (0.75 nmol P450/mg protein) are obtained by phenobarbital treatment. Immunoblotting with anti-P450 2B shows that the ratio of expressed 2B1 and 2B2 differs depending on the type of inducer. DEX predominantly leads to induction of 2B2, which may explain the low pentoxyresorufin O-depentylase activity in these microsomes.