Studies on the heterogeneity of the soluble chloroplast coupling factor 1: The formation of ε-deficient isozymes

Release of the chloroplast coupling factor 1 (CF 1 ) from thylakoid membranes by chloroform yields a heterogeneous population of isozymes. Following solubilization, subtle changes in the isozyme distribution occur. In particular, the percentage of e-deficient isozymes increases. Five parameters were evaluated for their role in the distribution of isozymes and net enzyme yield, (i) Extraction of spinach thylakoid membranes yields a five-subunit holoenzyme and derivative isozymes whereas Chlamydomonas reinhardtii membranes yield isozymes lacking a δ-subunit. (ii) Supplementing extraction buffers with MgCl 2 or CaCl 2 dramatically suppresses the release of total CF 1 , but does not appear to affect the distribution of isozymes. (iii) Loss of CF 1 by non-specific protein denaturation becomes detectable 24–48 h after enzyme solubilization. Analysis of CF 1 samples stored at room temperature for extended periods (up to 2 months) shows that the loss of solubility is uniform for the isozyme complex, rather than selective for agiven subunit. (iv) Proteolysis does not significantly affect the distribution of the e-subunit either before or after enzyme solubilization. (v) Dithiothreitol greatly enhances the formation of e-deficient isozymes from e-containing isozymes in both species studied and appears to account for the subtle changes observed following CF 1 solubilization.