Methanol-induced release of tightly bound adenine nucleotides from thylakoid-associated CF1.

Incubation of thylakoids in 33% methanol causes a release of the tightly bound nucleotides from CF1. This methanol effect is not a stimulation of nucleotide exchange, since no medium ATP or ADP is incorporated into CF1 during the methanol treatment. While the optimal conditions for stimulating the release of tightly bound ADP were similar to those for activating the ATPase, a direct relationship between the effects was not found. The tightly bound ADP does not represent a catalytic intermediate in this system, since (a) its rate of release is much slower than enzyme turnover, and (b) the substrate specificity for hydrolysis is different from that which promotes ADP release. A regulatory role for the tightly bound ADP in methanol-activated ATPase is also not indicated, since (a) activation of the ATPase occurs much more rapidly than ADP release, and (b) after the tightly bound ADP has been lost, high rates of ATP hydrolysis still require the presence of methanol, and (c) the small ATPase activity which persists after the removal of the methanol is not correlated with the loss of bound ADP. These results show that significant rates of ATP hydrolysis can occur with ADP still tightly bound to CF1. This argues against any model in which ADP regulates ATPase activity by binding directly to the catalytic site.