Fragmentation of chloroplast coupling factor in dependence of bound nucleotides Preparation of a reconstitutionally active form of subunit δ

AbstractPrevious studies on the ability of CF1, fragments to reconstitute photophosphorylation in CF1,-depleted thylakoids have shown that the degree of reconstitution was correlated with the presence of subunit δ in the fragment. This was taken as evidence that subunit δ was necessary for plugging the active proton channel CF0 [(1986) Eur. J. Biochem. 160, 635–643]. We questioned whether or not δ alone had this ability. In order to obtain δ we investigated the role of bound nucleotides in the stability of CF1. Starting from ammonium sulfate-precipitated CF1, we found that a low content of bound ADP (1 mol ADP/mol CF1) seemed to stabilize the β—δ interaction, while loosening the interaction between α,β and γ. By elution from an anion-exchange column in the presence of the nonionic surfactant Mega 9 we obtained β3δ and CF1(—δ) (both containing one ADP) or, after washing with alcohol/glycerol mixtures, β (nucleotide-free) and CF1/CF1(—ϵ). On the other hand, with a further 2 ADP and 2 ATP bound to CF1, (after incubation with excess ATP) the α-β-γ interaction was stabilized in such a way that subunit δ alone could be isolated from the complex. Subunit δ, when isolated by this procedure and added back to CF1-depleted thylakoids, reconstituted a high rate of photophosphorylation