Synthesis of adenosine triphosphate in respiration-inhibited submitochondrial particles induced by microsecond...

Phosphorylation of ADP to ATP was induced in nonrespiring submitochondrial particles (SMP) from rat liver by the application of electric pulses with field strengths of 10-35 kV/cm and a decay time of 60 mus. In all cases respiration was inhibited completely by using cyanide or rotenone. Newly formed ATP was measured by two independent methods, (i) the luciferase/luciferin bioluminescence assay and (ii) synthesis of [(32)P]ATP from ADP and (32)P(i). Both methods gave consistent and essentially identical results. Above 10 kV/cm the amount of ATP synthesized increased with increasing field strength, and at 30 kV/cm, approximately 40 pmol of ATP was synthesized per mg of SMP protein per pulse. ATP synthesis was shown to be related to the field-induced transmembrane potential, not to Joule heating of the suspension. Synthesis was abolished by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. The ionophores valinomycin and A23187 reduced the level of synthesis by 75% and 50%, respectively. ATP synthesis was also blocked by inhibitors of the F(0)F(1) ATPase complex, oligomycin, N,N'-dicyclohexyl carbodiimide, venturicidin, and aurovertin. The activities of the adenine nucleotide translocator and adenylate kinase, as well as release of bound nucleotides, could be excluded as sources of the new ATP. The data indicate that the minimal applied field at which ATP synthesis could be detected is approximately 8 kV/cm, corresponding to a maximal induced membrane potential of 60 mV in SMP. The maximal synthesis occurred at around 30 kV/cm, or an induced transmembrane potential of 200 mV. The duration of the applied pulse was also found to be critical, with 8 mus being the minimal triggering time for the synthesis. The induction of ATP synthesis in nonrespiring SMP by an externally applied electrical field is a direct demonstration of the transformation, by the mitochondrial inner membrane, of electrical energy into the chemical bond energy of ATP.