Oligomycin effects on ATPase and photophosphorylation of pea chloroplast thylakoid membranes

Abstract

Oligomycin inhibited the membrane-bound, Ca(2+)-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg(2+)-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.Sensitivity of isolated pea thylakoid Mg(2+)-dependent ATPase to oligomycin was found during summer months but not during the winter. This was traced to a seasonal variation in relative humidity with values of 20% or less during the winter, leading to lack of inhibition in the isolated chloroplasts. The effect of low relative humidity during growth could be reversed by placing plants for 17 to 20 hours in a chamber at 50% relative humidity. The optimum humidity range for sensitivity to oligomycin was between 40 and 60%.With chloroplasts potentially sensitive to oligomycin due to "permissive" growth conditions, the inhibition was modulated by a number of parameters during chloroplast activation or ATPase assay. Preincubation in high concentrations of KCl diminished oligomycin sensitivity, and this effect was reversed by valinomycin. The greatest sensitivity, as well as the highest ATPase rates, occurred only if thylakoids were activated by light and DTT at a relatively high temperature (35 to 40 degrees C). Controlling the levels of uncouplers and running the reaction in darkness rather than in the light, both of which diminished the degree of membrane energization, increased the sensitivity to oligomycin. It is possible that accessibility of the oligomycin binding site is diminished by membrane energization. These results are contrasted with those from studies of variability in sensitivity of yeast mitochondria to oligomycin.