valinomycin and 4-5-6-7-tetrachloro-2-trifluoromethylbenzimidazole

Application of an AC voltage to a model bilayer lipid membrane in the presence of electrogenic ionophores (valinomycin, protonophores CCCP and TTFB) was shown to change the steady-state current across the membrane induced by a DC voltage. The change in the DC current was proportional to the magnitude of the DC voltage and to the amplitude of the AC voltage and depended on both the AC frequency and the ionophore used. Application of this new experimental approach to the estimation of kinetic constants at individual stages of ionophore functioning is discussed.

Topics: Benzimidazoles; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Electric Conductivity; Electric Stimulation; Ionophores; Lipid Bilayers; Valinomycin

The fluorescent indicator pyranine was used for recording the internal pH of liposomes. The proton permeability was deduced from the velocity of the internal pH increase which was caused by shifting the external pH from 7 to 9. From valinomycin titration of the proton permeability in the presence of internal and external KCl (0.1 M), the permeability coefficient of H+ (PH) was obtained as 10(-4) cm/s at 22 degrees C. The coefficient was twice this value with the ATP synthase isolated from Wolinella succinogenes present in the liposomal membrane (10 mg protein/g phospholipid). ADP and phosphate had no effect on the latter PH. The protonophore TTFB (5 mumol/g phospholipid) increased the PH by 3 orders of magnitude. The permeability coefficients of H+ and K+ were used for calculating the delta uH and the proton flux associated with the phosphorylation which was driven by gradients of H+ and K+. For the conditions of limiting permeability of K+, the following conclusions were drawn. (1) In the steady state of rapid ion flux, the electrical potential across the liposomal membrane as calculated according to the Goldman equation, is directed opposite to the corresponding Nernst potential which is calculated from the K+ gradient. (2) The maximum turnover numbers of phosphorylation require a delta uH of 200-220 mV across the liposomal membrane. These values of delta uH and the corresponding turnover numbers are close to those brought about by the bacterial electron transport and the coupled phosphorylation. (3) The velocity of phosphorylation is linearly related to the proton flux. The slope of the line can be explained on the basis of an H+/ATP ratio of approx. 3.

Topics: Arylsulfonates; Bacteroidaceae; Benzimidazoles; Cell Membrane Permeability; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Phosphorylation; Potassium; Proton-Translocating ATPases; Protons; Spectrometry, Fluorescence; Uncoupling Agents; Valinomycin