Abstract
1. The synergistic effect of NADH on the NADPH-dependent mixed function oxidation of p-nitroanisole and hexobarbital can be measured both photometrically and by following the substrate-induced oxygen consumption. The increase in reaction rate is about 50% and lasts as long as NADH is present in the microsomal suspension.
2. The oxidation of added NADH is increased by hexobarbital, ethylmorphine and SKF 525-A. Lineweaver-Burk transformation of the NADH oxidation rates yields straight lines for xenobiotic substrates suggesting Michaelis constants similar to those obtained from metabolic experiments. NADH oxidation in the absence of NADPH is about half as rapid as in its presence.
3. Some substrates with spectral type II binding property do not affect the NADH oxidation, while p-aminophenol enhances it.
4. Metyrapone inhibits the hexobarbital-stimulated NADH oxidation non-competitively. Carbon monoxide, sulphydryl reagents and cyanide also inhibit it.
5. The results can be interpreted by assuming a reduced cytochrome P-450–substrate–oxygen complex which takes up a second electron preferentially from NADH.