[NADH- and NADPH-dependent formation of superoxide radicals in liver nuclei]

A method for the quantitation of the superoxide radical generation rate (V) in murine liver nuclei by the oxidation of 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine O2-. radicals with the formation of a stable nitroxyl radical recorded by the EPR method, has been developed. It was shown that NADP- and NADPH-dependent superoxide radical generation is suppressed by superoxide dismutase (approximately by 90%). The Km values for NADH and NADPH are 1.5 x 10(-6) and 4.4 x 10(-7) M, respectively; the maximal rate (0.2 nmol.min-1.mg protein-1) is equal for both substrates. Cyanide (greater than 2 mM) causes a practically complete inhibition of the O2-. generation by both substrates. It is suggested that there exists a single readily autooxidized site of O2-. generation by both substrates for NADH- and NADPH-dependent site of the electron transport chain in nuclear membranes.