Detection of ATP-dependent conformational change in the F1 portion and beta subunit of Escherichia coli H+-ATPase using 8-anilinonaphthalene-1-sulfonate.

The bindings of ATP to Escherichia coli coupling factor ATPase (F1) and its isolated beta subunit were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or the beta subunit, and this fluorescence was quenched on addition of ATP. Thus, ANS bound to the hydrophobic region of F1 or the beta subunit, and the binding of ATP was detected as a quenching of ANS fluorescence. The quenching of the fluorescence suggested that F1 or the beta subunit underwent a conformational change on binding to ATP. With and without ATP, 5.0 and 3.6 mol of ANS, respectively, bound to 1 mol of F1. On the other hand, with or without ATP about 1 mol of ANS bound to beta. The fluorescence quenching was specific for ATP and was not observed with GTP or CTP. It was dependent on pH, being higher at acidic pH, but it was not enhanced by MgCl2. The dissociation constants of F1 and the beta subunit for ATP were estimated to be 10(-4)-10(-5) M. The significance of these binding sites is discussed in relation to the mechanism of the ATPase.