| Abstract: | Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV. |
