A study on the mechanism of energy coupling in the redox chain

Generation of a membrane potential in the respiratory chain-deficient particles of beef heart mitochondria has been studied. For detection of membrane potential, phenyl dicarbaundecaborane (PCB^–,) and anilinonaphthalene sulphonate (ANS^–) probes were used. The respiratory chain-deficient submitochondrial particles were prepared after Arion and Racker (E-SMP), the procedure including complete disappearance of membrane structures and subsequent reconstitution of membrane vesicles as judged by the electron microscopy study. E-SMP were found to be deficient in cytochromesa,a ₃ and transhydrogenase, the cytochromeb,c ₁ andc content being lowered. Addition of NADH, succinate and tetramethyl-p-phenylenediamine+ascorbate did not induce either any oxygen consumption or membrane potential formation. Treatment of E-SMP with NADPH+NAD⁺ or with NADH+CoQ₀ did not entail generation of membrane potential, in contrast to that of parent, pyrophosphate submitochondrial particles (PP-SMP).E-SMP displayed an oligomycin-sensitive ATPase activity which could be increased by reconstitution of E-SMP with coupling factor F₁. Addition of ATP resulted in an uptake of PCB^– and enhancement of ANS^– fluorescence, the facts testifying to the formation of the membrane potential with plus inside E-SMP. Membrane potential formation was arrested by oligomycin, rutamycin, and uncouplers. Addition of respiratory chain inhibitors (antimycin+rotenone+ cyanide), complete reduction of respiratory carriers by dithionite and oxidation by ferricyanide were without effect on ATP-supported formation of membrane potential in E-SMP. It was concluded that utilization of ATP energy for the membrane potential generation does not depend on the state of the respiratory carriers and can be demonstrated under the conditions when none of redox chain coupling sites were functioning.Abbreviations PCB^– phenyl dicarbaundecaborane - ANS^– anilinonaphthalene sulfonate - E-SMP the respiratory chain-deficient submitochondrial particles - PP-SMP pyrophosphate submitochondrial particles