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.
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