Effect of protein-modifying reagents on ecto-apyrase from rat brain

We have tested several chemical modifiers to investigate which amino acid residues, present in the primary structure of the ecto-apyrase, could be involved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed in absence or the presence of the modifiers. Percentages of residual activity for ATPase and ADPase obtained when the following reagents were tested, are respectively: phenylglyoxal (an arginine group modifier) 17 and 30%; Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan group modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Otherwise, PMSF, a seryl protein modifier and DTNB, a SH-group modifier did not affect either ATPase or ADPase activity. Inhibitions observed after treatment with phenylglyoxal and Woodward's reagent were significantly prevented when the synaptosomal fraction was preincubated with ATP and ADP, indicating that the arginine and the side chain of glutamate or aspartate (carboxyl groups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates. Phenylglyoxal and Woodward's reagent also inhibited the GTPase and GDPase activities and this inhibition was prevented by preincubation with these substrates.