Identification of the Soluble Coupling Factor Transition in Calorimetric Scans of Chloroplast Membranes

Sensitive differential scanning calorimetry was employed to investigate the structure of spinach chloroplast membranes. In a relatively high ionic strength phosphate-buffered medium, major calorimetric transitions were resolved at 42 °C (A), 56. 5 °C (B), 69.5 °C (C), 76.5 °C (D), 82.7 °C (E), and 88.7 °C (F) (a lipid melting endotherm previously identified at ≈17 °C was not examined in this study). However, as both H+ and salt concentrations were lowered, the C endotherm was found to split into two component transitions: C₁ at 64.9 °C and C₂ at 69.6 °C. The C₁ transition was then demonstrated by five independent methods to derive from denaturation of the soluble subunit complex of the coupling factor (CF₁). Evidence for this conclusion was as follows: (i) Heat inactivation of CF₁ in situ occurred near the temperature of the C₁ transition, (ii) The endotherm of the isolated coupling factor (64.5 °C) was very similar to that of C₁ (64.9 °C). (iii) ADP and inorganic phosphate (two substrates of CF₁) both stabilized the C₁ and CF₁ denaturation endotherms by approximately the same amount, (iv) The denaturation temperature of the αand β subunits of CF₁ determined in intact chloroplast membranes was identical with the temperature of the C₁ transition, in both the presence and absence of ADP. (v) Elution of the soluble coupling factor from the membranes by three different methods removed the C₁ transition from the membrane scan.