ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.     

Investigation of the apparent inefficiency of the coupling between Photosystem II electron transfer and ATP formation

The maximum phosphorylation efficiency achieved with synchronous turnovers of Photosystem II (PS II) in spinach chloroplast lamellae is 0.3 molecules of ATP per pair of electrons transferred. This is the same as the efficiency observed for PS II operating alone in continuous light and would seem to indicate less than 50% coupling efficiency. Flash-induced ATP synthesis associated with both photosystems acting in unison closely approaches twice the flash-induced ATP synthesis associated with the Photosystem-I-dependent oxidation of duroquinol (itself 0.6) and comes close to equalling the highest efficiency observed in steady-state PS I + PS II electron transport. The anomalously low coupling efficiency seen when PS II is operating alone can be overcome by a ΔpH of two units imposed before flash illumination, or by a prior flash series involving the entire electron transfer chain. In contrast, prior electron transport through PS II alone is only slightly effective in enhancing the coupling efficiency of subsequent PS II turnovers. (It should be noted that in all cases where supplementary energy was provided, either by a proton gradient or by prior illumination, this supplementary energy was always below the energetic threshold for phosphorylation. Furthermore, the enhancement of PS II coupling efficiency by supplementary energy persisted even after a large number of subsequent PS II-inducing flashes). The efficiency of flash-induced ATP synthesis associated with whole-chain electron transfer or with PS-I-dependent duroquinol oxidation is also enhanced by the supplementary energy, but only during the first few inefficient flashes, suggesting that in this case the supplementary energy may simply be contributing to the initial build-up of an energetic threshold for ATP synthesis. This cannot be the case when the same supplementary energy contributes to the efficiency of the PS II reaction, since the enhancement then persists for a long time and contributes to an essentially constant flash yield of ATP. Our results imply that during electron transfer involving both photosystems, PS II participates in generating about half of the total ATP, whereas it operates inefficiently only when operating alone. Since hydrogen ions produced by PS I are able to raise the efficiency of subsequent PS-II-dependent phosphorylation, at least some cooperation between the two photosystems takes place and this suggests some donation of protons from PS I to PS II. However, the inability of PS II alone to achieve high efficiency, even with prolonged pre-illumination, would seem to indicate some functional distinction of protons from the two photosystems.