Ionic composition of the medium, surface potential and affinity of the membrane-bound chloroplast ATPase for its charged substrate ADP

Abstract

Because the membrane-bound ATPase and its substrate ADP are both electronegative, the effect of the ionic concentration on the kinetic parameters of photophosphorylation V max and K m (ADP) was examined in lettuce thylakoids in various conditions. The initial rate of ATP synthesis was measured by the “scalar” H + consumption in the external medium, in conditions where the “vectorial” electrochemical proton gradient (restricted here to ΔpH) was maintained constant; ΔpH was estimated from the 9-aminoacridine fluorescence quenching. This signal was calibrated by experiments where the “phosphate potential” Δ G p was in equilibrium with ΔpH. With a fixed H +/P stoichiometry of 3, a strong dependence of the probe response on the ionic conditions was found. On the other hand, at a given ΔpH, the K m for ADP was unexpectedly found insensitive to the ionic conditions. Especially, lowering the Mg 2+ concentration from 5 mM to 0.5 mM diminished V max considerably but did not affect K m. Attempts to measure the membrane surface electrical potential ψ s by salt-induced release of 9-aminoacridine and by particle electrophoresis showed important discrepancies between the two methods. This seems not to be due to the difference between the potential measured at the membrane surface, more precisely at the Stern or Gouy levels, and that at the plane of shear only. It is more likely a consequence of side effects with 9-aminoacridine. Taking the electrokinetic potential ζ as representative of ψ s, it is then shown that the membrane surface potential is already small in the minimum salt medium (ca. − 15 mV) and, consequently, that the ADP concentration near the membrane, and hence K m, can barely change with the ionic concentration increase. This makes that the K m determinations, based on ADP concentrations in the bulk phase, are close to the true K m, which must take into account the actual ADP concentration at the membrane level.