Heterogeneity of ATP-hydrolyzing sites on reconstituted CF0F1

AbstractThe proton translocating ATP-synthase from chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. [γ-32P]ATP hydrolysis was measured under uni-site conditions. When 1 mM unlabeled ATP was added so that all ATP-binding sites were occupied, [γ-32P]ATP bound to the first site, was hydrolyzed with a rate of 0.5 ATP/(CF0F1 s). In a second experiment, first cold ATP was hydrolyzed under uni-site conditions and then 1 mM [γ-32P]ATP was added. This allows under otherwise identical conditions the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. It resulted in a rate of 80 ATP/(CF0F1 s). It is concluded that the catalytic nucleotide binding sites are heterogeneous: there exists one nucleotide binding site which hydrolyzes ATP with a maximal turnover of 0.5/s and another one (or two) which hydrolyze ATP with a turnover of 80/s. The latter one is the catalytic site for maximal turnover