Identification of Chloroplast Coupling Factor by Freeze-etching and Negative-staining Techniques

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.