PENTAGON Mitochondrial ATPASE from beef heart (Pulsed EMF)

PENTAGON Mitochondrial ATPASE from beef heart (Pulsed EMF)

Witt and co-workers [28] were the first to report activation of ATP synthesis of chloroplasts by pulsed electric fields. The yield of ATP was less than one per enzyme per electric pulse and it was assumed that the electric field triggered the release of tightly bound nucleotides from the enzyme surface. Our experiments were performed with beef heart mitochondrial ATPase and our goal was to demonstrate that a single pulse of electric field can induce enzyme turnover [29-33]. The electron transport activity of submitchondrial particles was inhibited by rotenone or cyanide or both.

Thus, the energy source for ATP synthesis, about 42 kJ/mol, must be derived from the applied electric field. Using the capacitor discharge technique it was shown that ATP was formed from ADP and P(i) and this synthesis was inhibited completely by oligomycin and FCCP (carbonyl cyanide p-triflouromethoxyphenol- hydrazone), the former being a potent inhibitor and the latter an ionophore of proton. When dithiothreitol was present in the medium, as much as 5 ATP molecules per enzyme were generated with a single pulse of 30 kV/cm (300 mV of transmembrane potential) with a decay constant of 100 us. We interpret these results as due to the oxidation-reduction cycle of certain SH groups in the ATP synthetic process. Firgure 5A shows some results of these experiments using the exponentially decaying electric field.

Alternating electric fields have also been used to demonstrate ATP synthesis. In most experiments, we used an electric field of 60 V/cm at various frequencies. The ATP yield per ac cycle is low (approximately 10^-4) because of the low energy level of the applied field. However, the experiment is simple and the results are essential for clarifying certain concepts of the ECC model, as discussed below.

Another interesting observation is that the natural inhibitor peptide of the ATPase has a profound effect on the electric field induced ATP synthesis. Synthesis was effective only if the peptide was removed. Figure 5B gives some of the results which show an increase in ATP yield as the natural inhibitor peptide dissociates with time from the enzyme.

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