ATP-diphosphohydrolase activity in rat renal microvillar membranes and vascular tissue

Abstract

Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations, The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney, ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to 'right side out' membranes, were characterized, The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney, ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties, These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap(5)A. The M(r) of native ATPase and ADPase activities,vas determined by the Co-60 irradiation-inactivation technique being around 65 kDa for both hydrolytic activities, Immunowestern blot analysis also supports the presence of apyrase in microvilli, Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.