Identification of brain ecto-apyrase as a phosphoprotein

Ecto-apyrase is a transmembrane glycoprotein that hydrolyzes extracellular nucleoside tri- or diphosphates. Apyrase activity is affected by several physiological and pathological conditions indicating the existence of regulatory mechanisms. Considering that apyrase presents consensus phosphorylation sites, we studied the phosphorylation of this enzyme. We found an overlay of the immunoblotting and phosphorylated bands in three different preparations from rat brain: (a) hippocampal slices, (b) synaptic plasma membrane fragments and (c) cultured astrocytes. In addition, two-dimensional electrophoresis separations with human astrocytoma cells were done to identify unequivocally the coincidence between the immunodetected and phosphorylated protein. These observations indicate that apyrase can be detected as a phosphoprotein, with obvious implications in the regulation of this enzyme.