Electrochemical proton gradient across the cell membrane of Halobacterium halobium: effect of N,N'-dicyclohexylcarbodiimide, relation to intracellular adenosine triphosphate, adenosine diphosphate, and phosphate concentration, and influence of the potassium gradient

Abstract

The proton motive force across the cell membrane of halobacterial cells was estimated and compared to intracellular values of ATP, ADP and Pi concentrations with respect to the chemiosmotic hypothesis. The accumulation of 14C-labeled indicator substances, triphenylmethylphosphonium for the membrane potential and 5,5-dimethyloxazolidine-2,4-dione for the pH difference between the cell interior and the medium, was measured in the cells. Values up to 270 mV for the proton motive force were found in cells pretreated with N,N''-dicyclohexylcarbodiimide (DCCD, 10-4 M, 30.degree. C, 12 h). Upon illumination a high membrane potential is generated, which is then gradually replaced by a large pH difference. Cells treated with lower DCCD concentration show only an enhancement of membrane potential upon illumination; the pH difference remains at a low level. Under anaerobic dark conditions, untreated cells maintain a proton motive force of 120-140 mV, which is equilibrated with the intracellular levels of ATP, ADP and Pi. The pH gradient is 1 unit at pH 6 but 0 at pH 8. The membrane potential is low (60-80 mV) at pH 6 and high (120-130 mV) at pH 8. The proton translocating ATPase compensates for the lowered pH difference at high external pH values by enhancing the membrane potential. The concentration difference of the K+ influences the proton motive force and the intracellular ATP levels, apparently via its action on the membrane potential. When the difference of the chemical potential of the K+, expressed in millivolts, exceeds the preexisting membrane potential, the intracellular ATP level is enhanced. When the difference of the chemical potential of the K+ (millivolts) is smaller than the membrane potential, the ATP level is decreased.