title: Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles.
abstract: Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.
authors: Department of Biology, Technion-Israel Institute of Technology, Haifa.
authors: G D
authors: GD
authors: Eytan
authors: E
authors: E
authors: Carlenor
authors: J
authors: J
authors: Rydström
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000609:Aminoacridines; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials / Q000187:drug effects; D008929:Mitochondria, Heart / Q000201:enzymology*; D009550:Nigericin / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D014634:Valinomycin / Q000494:pharmacology*
chemical_list: D000609:Aminoacridines; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008081:Liposomes; D014634:Valinomycin; C010847:9-amino-6-chloro-2-methoxyacridine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009550:Nigericin