Study on Mechanisms of ATP Synthesis in Single Mitochondria

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The mitochondrial activities related to ATP production were measured in single mitochondria with fluorescence microscopy. So far, studies on mitochondria have been performed with mitochondrial suspension. Therefore, it has been impossible to detect asynchronous phenomena like the opening and the closing of a channel. In the present study, we developed the technique to measure the activity of single mitochondria to detect the phenomena that has not been detected with mitochondria suspension.To observe single mitochondria with fluorescence microscopy, mitochondria were adsorbed on the cover slip coated with polyphenolic protein. Since the adsorption did not decrease the ATP production rate in mitochondria, this method for adsorption was elucidated to be useful for the measurements of mitochondrial activity. Upon addition of malate as a respiratory substrate, the time-courses of NADH formation and Flavin reduction in single mitochondria was similar to the results obtained for mitochondrial suspension. On the other hand, mitochondria showed the generation and disruption of the membrane potential due to the opening and the closing of the permeability transition pore. The opening and the closing depends on the redox state of the electron transport chain.