Histochemical and Biochemical Studies on the Respiratory Enzymes Part 1. The Reduction Mechanism of Tetrazolium Salts by the Respiratory Enzyme System

With the purpose to establish the integrating methods for the selective histochemical demonstration and colorimetric estimation of each enzyme and enzyme system belonging to the terminal respiratory enzyme system, an alytical study on the reduction of neotetrazolium chloride by tissue homogenates was carried out in the present report using various substrates and inhibitory agents, and the steps with which the reaction of neotetrazolium chloride reduction conjugates were determined as follows. 1. The reaction of neotetrazolium reduction by tissue homogenetes using sodium succinate as substrate is not the succinic dehydrogenase reaction but the succinoxidase system reaction; and the reaction takes place cojugating about 50 per cent in the step of the succinic dehydrogenase system, of these about 15 per cent conjugates in the step prior to the antimycin A sensitive step and 35 per cent in the step itself; and about 50 per cent in the step of cytochrome c oxidase. 2. In the case using p-phenylenediamine as substrate the reaction of neotetrazolium reduction is the reaction due to the activity of cytochrome c-cytochrome oxidase system, and when p-phenylenediamine is used with the sufficient amount of cytochrome c, the reaction appears to be dependent on cytochrome c oxidase activity. Neotetrazolium reduction in all these reactions takes place conjugating in the step of cytochrome c oxidase. 3. In the case where DPN and substrates taking DPN as a coenzyme are used, the reaction of neotetrazolium reduction is mainly the reaction conjugating at the step below antimycin A sensitive step in the DPNH-cytochrome c reductase system. 4. Endogenous dehydrogenase reactions are the sum total reactions conjugating at the steps prior to the antimycin A sensitive step in the terminal electron transport system and with other various reduction systems which are not inhibited by antimycin A.