QUANTITATIVE CYTOCHEMICAL STUDIES OF STIMULATION OF RAT-LIVER SUCCINATE-NEOTETRAZOLIUM REDUCTASE BY PHENAZINE METHOSULPHATE AND QUINONES AND OF INHIBITION BY NEOTETRAZOLIUM WITH OBSERVATIONS ON ALTERED FORMAZAN LOCALISATION

The succinate-neotetrazolium reductase system has been studied in sections of suitably frozen rat liver with respect to the concentration of succinate, and with reference to substances which increase the rate of enzymic activity. The effect of these substances in overcoming the inhibitory action of neotetrazolium on the enzyme complex at elevated tetrazole concentrations has also been studied. N-Methyl phenazonium methosulphate (PMS) produced an approximately tenfold increase in the rate of reaction. Half-maximal activity was obtained in a range of succinate concentration from 0.5-1.0 × 10-4 M. No value for the Km could be obtained graphically from double reciprocal plots of enzyme velocity and substrate concentration, as the graphs were curved. Although menadione stimulated enzymic activity, the inhibitory action of elevated concentrations of neotetrazolium on succinate-neotetrazolium reductase was not overcome. Ubiquinone30 not only stimulated enzymic activity, but also raised the optimum level of neotetrazolium threefold. The Michaelis constant for neotetrazolium-reductase was altered slightly by the introduction of ubiquinone or PMS into the reaction system. Elevated levels of neotetrazolium inhibit the enzyme complex apparently by interference with quinone function. The characteristic pattern of formazan deposition in the liver lobule was significantly changed in the presence of added ubiquinone or PMS, but not menadione. The distributions of the primary flavoprotein dehydrogenase and of endogenous quinone in the liver lobule appeared to be different. The pH optimum and the neotetrazolium concentration which is optimal for enzymic activity were found to be interdependent; elevated concentrations of neotetrazolium lowered the pH optimum, while an increase in pH lowered the optimal concentration of the tetrazole. © 1968.