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A study was made of the interaction of the negative (−127…−160) and positive (−69…−98)cis-acting elements of the minimal promoter of the cytochrome P4502B2 gene with nuclear proteins of rats injected with phenobarbital and triphenyldioxane. On evidence of the gel retardation assay, binding of nuclear proteins to the negative element is suppressed, and that to the positive one is enhanced 6–9 h after injection. Binding to both elements is maximal 18–48 h after injection and decreases to the control level 72 h after injection. It is assumed that, when derepressed, the negative element acts as a positive one. Thein vitrobinding proved to correlate with changes in theCYP2B1/2mRNA content. The data obtained with triphenyldioxane testify to the involvement of the proximal promoter in the inducer-dependent activation of cytochrome P4502B2.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the ‘binding protein’ for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.

In rat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (O2-), whereas cytochrome c, 2,6-dichlorophenol-indophenol and ferricyanide accepted electrons from NADPH-specific flavoprotein only directly. Nitro Blue Tetrazolium was reduced both by O2- and by the direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2--dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of O2- in rat liver microsomal fraction were determined.