Preparation of the epsilon subunit and epsilon subunit-deficient chloroplast coupling factor 1 in reconstitutively active forms

Abstract

A simple and rapid method is described for releasing the epsilon subunit from chloroplast coupling factor 1 by treatment with 20% ethanol on an anion exchange column. The resulting epsilon subunit-deficient enzyme is a permanently active Ca2+-ATPase, but an inactive coupling factor. Recombination with the epsilon subunit returns the enzyme to the latent Ca2+-ATPase state and restores its ability to synthesize ATP when reconstituted with thylakoid membranes. The epsilon subunit is not required for binding coupling factor 1 to the membrane, but its presence is necessary to prevent the leak of protons through the hydrophobic portion of the coupling factor complex.