THE EFFECT OF ENERGY-TRANSFER INHIBITORS ON THE PHOTOPHOSPHORYLATION PARAMETERS IN LETTUCE THYLAKOIDS AND THE QUESTION OF THE KM (ADP) VARIATION WITH MEMBRANE ENERGIZATION

In lettuce thylakoids illuminated with continuous light, buffering of ΔpH variations by internal accumulation of hexylamine was used to measure the initial rate of photophosphorylation upon ADP addition, keeping ΔpH constant whilst ADP concentration was changed (variable vectorial H+ efflux). Then, the ΔpH was adjusted to another, constant, value and a similar concentration curve was traced. At high ΔpH (above 3.5), the apparent Michaelis constant for ADP was found between 15 and 40 μM, increasing with the proton gradient. Tentoxin, an irreversible F1 inhibitor, did not change this apparent affinity for ADP, whereas phloridzin, a reversible F1 inhibitor, lowered the Km. DCCD and venturicidin, two F0 inhibitors, unexpectedly decreased the Km for ADP. These results are compatible in principle with the existence of a diffusion barrier in the unstirred layer covering the membrane, which limits the access of ADP to ATP-synthases at high turnover rates. This would lower the actual ADP concentration around the enzymes and raise the apparent Km, which is based on concentration known in the bulk phase. However, considering the quantitative effects observed, an additional hypothesis is that the protonic activation of the enzyme is not, as usually believed, an all-or-nothing process, but involves different functional states with different affinities for the substrate ADP. © 1990.