Repeated extractions of spinach thylakoid membranes with a solution containing 50 mM sodium cholate, 1 M NaCl, 3 mM MgCl2, 0.2 M sucrose and 20 mM tricine at pH 8.4 for 15 min perfectly inhibited the O2 evolution of the thylakoids, concomitant with a complete release of the 17- and 23-kDa proteins and partial release of many other proteins from the thylakoid membranes. Recovery of O2 evolution in the cholate-treated thylakoids was achieved up to about 40% of that in the original thylakoids by the simultaneous reinsertion of the 17- and 23-kDa proteins, but not by the reinsertion of one of them only. The recovery of O2 evolution induced by the reinsertion of the 17- and 23-kDa proteins was enhanced by the further addition of a certain fraction of the crude thylakoid extract up to about 70% of the non-depleted control, suggesting that in addition to the 17- and 23-kDa proteins, one or more unknown component(s) released partially from the thylakoids upon cholate treatment is (are) also (a) constituent(s) of the O2 evolving apparatus. The purified 34-kDa protein did not replace the unknown component.
