The ATPase activity of the F1moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 °C in the presence of MgCl2. The concentration of MgCl2(130 μM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1complex of a subunits and part of the 7-subunit population. The latter form a precipitate while the β, δ, and ∊ subunits, and the remaning part of the 7-subunit population, remain soluble. Dissociation is not a sudden “all or none” event but parallels loss of ATPase activity until a subunits have almost completely dissociated together with about 50% of the 7-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPiat high concentration (>200 mM) is effective in partially protecting F1against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0moiety in F1-depleted particles. When bound to F0, F1is protected completely against divalent cation induced inactivation. These results indicate that F1contains, in addition to the high-affinity divalent cation sites involved in promoting catalysis, additional sites of lower affinity which either are masked in the intact ATP synthase complex (f0f1)or interact with the intact complex with a functional purpose other than inactivation.
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