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

We have tested several chemical modifiers to investigate which amino acid r esidues, present in the primary structure of the ecto-apyrase, could be inv olved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed-in absence or the pres ence of the modifiers. Percentages of residual activity for ATPase and ADPa se obtained: when the following reagents were tested, are respectively: phe nylglyoxal (an arginine group modifier) 17 and 30%;: Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan gro up modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Othe rwise. PMSF, a seryl protein modifier and DTNB, a SH-group modifier,did not affect either ATPase or ADPase activity. Inhibitions observed after treatm ent 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 g roups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates; Phsnylgl yoxal and Woodward's reagent also inhibited the GTPase and GDPase activitie s and this inhibition was prevented by preincubation with these substrates: (C) 1999 Elsevier Science Ltd. All rights reserved.