Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells

(1) Photophosphorylation, fCa²⁺-ATPase and Mg²⁺-ATPase activities of isolated chloroplasts were inhibited 55–65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (ψ_w) of ?25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves.(2) These losses in activity were reflected in coupling factor (CF₁) isolated from the leaves, since CF₁ from leaves with low ?_w had less Ca²⁺-ATPase activity than control CF₁ and did not recouple phosphorylation in CF₁-deficient chloroplasts. In contrast, CF₁ from leaves having high ?_w only partially recoupled phosphorylation by CF₁-deficient chloroplasts from leaves having low ?_w. This indicated that low ?_w affected chloroplast membranes as well as CF₁ itself.(3) Coupling factor from leaves having low ψ_w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF₁ from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF₁ from leaves having low ?_w differed from control CF₁. The CF₁ from leaves having low ?_w also had decreased ability to bind fluorescent nucleotides (?-ATP and ?-ADP).(4) Exposure of isolated CF₁ to low ?_w in vitro by preincubation in sucrose-containing media inhibited the Ca²⁺-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg²⁺ in the preincubation medium markedly inhibited Ca²⁺-ATPase activity.(5) These results show that CF₁ undergoes changes in cells which alter its phosphorylating ability. Since low cell ?_w changed the spectroscopic properties but not other protein properties of CF₁, the changes were most likely caused by altered conformation of the protein. This decreased the binding of nucleotides and, in turn, photophosphorylation. The inhibition of ATPase activity in CF₁ in vitro at low ?_w and high ion concentration mimicked the change in activity seen in vivo.