The transmembrane electrical potential in Rhodopseudomonas sphaeroides determined from the distribution of tetraphenylphosphonium after correction for its binding to cell components.

Abstract

The membrane potential was determined in intact cells of Rhodopseudomonas sphaeroides from the distribution of the lipophilic cation tetraphenylphosphonium (Ph4P+) after correction for probe binding to cell components. The concentration of Ph4P+ in the external medium of the cells was recorded with a Ph4P+ ‐selective electrode. Probe concentrations up to 20 μM have no effect on the membrane potential. The binding of the probe to the cells is proportional to the free probe concentration up to 80 μM and increases with the pH of the medium. The binding characteristics were determined for the different cell components: the cytoplasm, the cytoplasmic membrane and the outer membrane (and cell wall). The results indicate that the binding sites are for 47% located at the inside of the cytoplasmic membrane and for 53% at the outside of the cytoplasmic membrane. With the equation derived previously [J. S. Lolkema, K. J. Hellingwef, W. N. Konings, Biochim. Biophys. Acta, 681 (1982) 85–94] the membrane potential can be calculated after correction for probe binding. These membrane potentials were 58 mV lower than the uncorrected values. In the dark the membrane potential is – 68 mV. Upon illumination of the cells the membrane potential increases to – 97 mV if nigericin is present. In the absence of nigericin a rapid conversion of the membrane potential into a pH gradient occurs and steady state membrane potentials are generated which are only slightly higher than the membrane potentials found in the dark.