CONFORMATION AND ACTIVITY OF CHLOROPLAST COUPLING FACTOR EXPOSED TO LOW CHEMICAL-POTENTIAL OF WATER IN CELLS

Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated [spinach (Spinacia oleracea)] chloroplasts were inhibited 55-65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (.PSI.w) of -25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low .PSI.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 .PSI.w only partially recoupled phosphorylation by CF1-deficient chloroplasts from leaves having low .PSI.w. Low .PSI.w apparently affected chloroplast membranes as well as CF1 itself. CF1 from leaves having low .PSI.w had the same number of subunits, the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, UV absorption and ciruclar dichroism showed that CF1 from leaves having low .PSI.w differed from control CF1. The CF1 from leaves having low .PSI.w also had decreased ability to bind fluorescent nucleotides (.epsilon.-ATP and .epsilon.-ADP). Exposure of isolated CF1 to low .PSI.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. These results show that CF1 undergoes changes in cells which alter its phosphorylating ability. Since low cell .PSI.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 .PSI.w and high ion concentration mimicked the change in activity seen in vivo.