Nitrite binding to rabbit liver microsomes and effects on aminopyrine demethylation.

Sodium nitrite was examined for its ability to interact with liver microsomes from phenobarbitaltreated rabbits. In dithionite- or NADPH-reduced microsomes, nitrite rapidly produced a difference spectrum with α, β and Soret peaks at 586, 510, and 446 nm, respectively, with a large trough at 417 nm. The Soret peak diminished with time as the trough deepened and shifted to 429 nm. Concomitant with the development of the difference spectrum was a decrease in the ability of reduced cytochrome P-450 to bind carbon monoxide. When preincubated with NADPH-reduced microsomes under anaerobic conditions, nitrite behaved as a noncompetitive inhibitor of aminopyrine demethylase activity. Inhibition kinetics revealed two components with apparent inhibition constants of 0.2 and 31 mM nitrite. Maximum inhibition of aminopyrine demethylase by nitrite was obtained only after preincubation under anaerobic and reducing conditions. The rate and extent of inhibition were increased by decreasing the pH of the medium during preincubation. Inhibition of aminopyrine demethylation by nitrite does not appear to be mediated by oxidation effects of nitrite on lipids or essential sulfhydryl groups of eytochrome P-450. The evidence suggests that nitrite or a reduction product of nitrite, such as nitric oxide, binds tightly to reduced cytochrome P-450 to prevent carbon monoxide binding and inhibit aminopyrine demethylation activity.