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

M L Richter,W J Patrie,R E Mccarty

doi:10.1016/s0021-9258(17)42797-9

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.

Highlights

Reconstitutively Active Forms*SpinachCF, was prepared by amodification [4] of previously reported [8, 9] methods. Spinach chloroplast thylakoidwsere freed of (Received for publication, March 12, 1984) CF, by treatment with 2 M NaBr [10]
Thecolumn was washed with 20 ml of the samebuffer, and thee subunit was eluted by washing with 50-100 mI of 25 mM Tris-HC1 containing 2 mM ATP, 5 mM
A simple and rapid method is described for releasing the E subunit from chloroplast coupling factor 1 by treatment with 20% ethanol on an anion exchange column

Summary

Reconstitutively Active Forms*

SpinachCF, was prepared by amodification [4] of previously reported [8, 9] methods. Spinach chloroplast thylakoidwsere freed of (Received for publication, March 12, 1984) CF, by treatment with 2 M NaBr [10]. Purified 6 subunit and e-deficient CF, were obtained by binding CF, (10-30 mg) to a column (1 X 10 cm) of DEAE-cellulose equilibrated at room temperature with 25 mM Tris-HCI (pH7.9) containing 2 mM ATP and mM dithiothreitol. Thecolumn was washed with 20 ml of the samebuffer, and thee subunit was eluted by washing with 50-100 mI of 25 mM Tris-HC1 (pH7.9) containing 2 mM ATP, 5 mM dithiothreitol, 30% (v/v) glycerol, and 20% (v/v) ethanol. A simple and rapid method is described for releasing the E subunit from chloroplast coupling factor 1 by treatment with 20% ethanol on an anion exchange column. Ca'+-ATPase activity was measured at 37 "C in thepresence of 50 mM Tris-HC1 (pH 8.0), mM ATP, and 5 mM CaC1'. Membrane.Isolated CF,' is a latentATPasethat can be activated by a number of treatments, including heat, dithio-

RESULTS

Only t subunit

Findings

DISCUSSION