Freezing of isolated thylakoid membranes in complex media : III. Differences in the pattern of inactivation of photosynthetic reactions.

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO3 and 20 mM K2SO4 in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H(+) gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H(+) permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF1). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N'-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF1 remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF1 and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.