Investigations on nucleotide binding sites of isolated chloroplast ATPase by modification with 7-chloro-4-nitrobenzofurazan

Abstract

Addition of NBD-Cl to isolated and nucleotide-depleted CF 1 at pH 7.5 leads to binding of 2 NBD molecules on tyrosines of CF 1, one in one of the 3 α-subunits and one in one of the 3 β-subunits. MgADP reduces the amount of bound NBD in β, but not in a. Modification as a function of time is biphasic, showing an initial rapid and a slow kinetic component. MgATP represses the initial rapid phase of binding. Incubation of O-Tyr-NBD-CF 1 at pH 9 causes a time-dependent shift of the NBD-molecule from tyrosine to lysine. The O-Tyr-NBD-formation is an obligatory intermediate for specific N-Lys-NBD-formation. ADP reduces the formation of the lysine-bound NBD drastically.