Neotetrazolium salt (NT) as an electron acceptor in living cell: a study on the JTC-3 and HeLa cells in culture

1. By using a monolayer of tissue culture cells, JTC-3, JTC-3', JTC-3" and HeLa cells, the assays of neotetrazolium (NT) reduction at the cell level have been conducted on the cells without detaching the cells from the culture glass vessel wall. 2. It has been found that the activity of endogenous NT reductase is extremely high in the living tissue culture cells. The endogenous NT reductase activity is found to be in the descending order of HeLa > JTC-3' > JTC-3 > JTC-3". The endogenous NT reductase activity increased in the medium having a low level of proteins and decreased in the absence of yeast extract. 3. It has been demonstrated that most of the endogenous NT reduction in the JTC-3 cell groups takes place at the step of flavoprotein in the NADHdiaphorase system, and a portion of it occurs being coupled with the ubiquinone step. In contrast, in HeLa cell it is p:>stulated that aside from the NADH-diaphorase system, succinoxidase system is involved in this reaction. 4. The coupling site of the succinate NT reductase system with the terminal electron transport system in the JTC-3 cell groups also differs from that in HeLa cells. Namely, in JTC-3 cell groups about 50% of the coupling occurs at the step later than antimycin A sensitive step and the remaining 50 % most likely at the ubiquinone step~ whereas in the HeLa cell most of the coupling takes place at the step later than antimycin A sensitive step. 5. Although there can be observed variations in the activity of the succinate NT reductase, hardly any difference was observed among the JTC-3 cell groups in the rate of the reduced NT amounts at several coupling sites, revealing that the change in the composition of the culture medium does not bring about any essential change in the coupling sites and their mutual activity rates of succinate NT reductase system. 6. Both endogenous NT reductase activity and succinate NT reductase activity have been accelerated markedly by potassium cyanide. The result indicates that in living cell the electrons are not transferred to NT at the level of cytochrome oxidase and O2.