Inactivation of ecto-ATPase activity of rat brain synaptosomes

The ecto-ATPase activity of synaptosomes plasma membrane decays exponentially as a function of time from 0.35 +/- 0.05 to 0.08 +/- 0.02 mumol ATP hydrolyzed per min per mg synaptosome protein. The first-order rate constant of inactivation is dependent on the Mg-ATP concentration varying from 0.042 +/- 0.001 min-1 with 30 microM ATP up to 0.216 +/- 0.003 min-1 with 2 mM ATP. The non-hydrolyzable ATP analogue, beta-gamma-methyleneadenosine 5'-triphosphate, did not produce inactivation of the ecto-ATPase activity. Thus, the inactivation of the ecto-ATPase activity requires hydrolysis of ATP. Product inhibition can be excluded because ADP, AMP, adenosine and inorganic phosphate up to 1 mM had no effect on the inactivation of the ecto-ATPase. Concanavalin A partially protected against the ATP-dependent inactivation. The ecto-ATPase inactivation produced by Mg-ATP is partially reverted by centrifugation, removal of the supernatant and resuspension of synaptosomes in a fresh medium. This partial reversion occurs in parallel to the release to the supernatant of phophorylated protein(s) of 90-95 kDa. Alkaline phosphatase treatment fully reverts the ecto-ATPase inactivation. We conclude that the ATP-induced inactivation is mediated, at least partially, by phosphorylation of membrane proteins.