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 ph ysiological and pathological conditions indicating the existence of regulat ory 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 preparati ons from rat brain: (a) hippocampal slices, (b) synaptic plasma membrane fr agments and (c) cultured astrocytes. In addition, two-dimensional electroph oresis separations with human astrocytoma cells were done to identify unequ ivocally the coincidence between the immunodetected and phosphorylated prot ein. These observations indicate that apyrase can be detected as a phosphop rotein, with obvious implications in the regulation of this enzyme.