A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (MI 17000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25qo glycerol to reactivate oxygen evolution. Pigment system 2 Reconstitution of oxygen evolution Reconstituted membrane Spinach chloroplast Thylakoid membraneVesicle

Repeated extractions of spinach thylakoid membranes with a solution containing 50 mM sodium cholate, 1 M NaCI, 3 mM MgClz, 0.2 M sucrose and 20 mM tricine at pH 8.4 for 15 min perfectly inhibited the 02 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 02 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 02 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 nondepleted 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 02 evolving apparatus. The purified 34-kDa protein did not replace the unknown component.

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2016-11-02T23:50:20