The binding of eosin-labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution

AbstractWe investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF1 (containing δ) and of CF1(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF1 decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF1) relative to δ (free). CF1(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (Kd ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF1, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF1