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.
Martín-Romero
F J
FJ
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
García-Martín
E
E
Gutiérrez-Merino
C
C
D000241:Adenosine / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000201:enzymology*
D004791:Enzyme Inhibitors; D010710:Phosphates; D003208:Concanavalin A; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C027201:ectoATPase; D000241:Adenosine