Ecto-ATPase and Ecto-ATP-diphosphohydrolase Are Co-localized in Rat Hippocampal and Caudate Nucleus Synaptic Plasma Membranes

N. NEDELJKOVIC¹, A. BANJAC², A. HORVAT², M. STOJILJKOVIC¹, G. NIKEZIC²

¹Department of General Physiology and Biophysics, Faculty of Biology, University of Belgrade, ²Laboratory of Molecular Biology and Endocrinology, Vinca Institute of Nuclear Sciences, Belgrade, Yugoslavia

Received August 20, 2002
Accepted February 7, 2003

Summary
Enzymes that hydrolyze extracellular ATP, i.e. ecto-ATPase and ecto-ATP diphosphohydrolase (ATPDase), can be differentiated by ability of the latter to hydrolyze ADP and by slightly different kinetic properties of the two enzymes. Synaptic plasma membrane fractions isolated from rat hippocampus and caudate nucleus exhibit ADP-hydrolyzing activity, as revealed by the enzyme assay, and the presence of ecto-ATPase protein, as revealed by immunological identification on Western blot. These findings indicate that both enzymes are co-expressed in the synaptic membrane compartment of hippocampal and caudate nucleus neurons. Kinetic analysis was performed to determine the relative contribution of each enzyme to the total ATP-hydrolyzing activity, while an inhibition study was carried out in order to exclude the interference of other nonspecific ATPase and phosphatase activities. Based on the kinetic properties, sensitivity to inhibitors and VATP/VADP ratio of about 2, we concluded that a substantial portion of ATP-hydrolyzing activity in both synaptic membrane preparations can be ascribed to the catalytic action of ATPDase. On the other hand, the highest catalytic efficacy when ATP is the substrate and the greater abundance of ecto-ATPase protein in caudate nucleus preparation suggest that the relative contribution of ecto-ATPase to the total ATP-hydrolyzing activity in the caudate nucleus is higher than in the hippocampus.

Key words
Ecto-ATPase • Ecto-ATPhydroxylase • Hippocampus • Caudate nucleus • Rat