Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol

Abstract

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F 1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In sosuble F 1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F 1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F 1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP on ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.