Prevention and recovery of CuSO<inf>4</inf>-induced inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase in rat brain synaptosomes by EDTA

Abstract

Enzymatic activities of Na+/K+-ATPase and Mg2+-ATPase from rat brain synaptic plasma membrane were studied in the absence and presence of EDTA. The aim of the study was to examine the ability of this strong chelator to prevent and recover the CuSO4-induced inhibition. The influence of experimentally added CuSO4 and EDTA on MgATP2- complex and 'free' Cu2+ concentrations in the reaction mixture was calculated and discussed. CuSO4 induced dose-dependent inhibition of both enzymes in the absence and presence of 1 mM EDTA. In the absence of EDTA, the IC50 values of Cu2+, as calculated from the experimental curves, were 5.9 x 10-7 M for Na+/K+- ATPase and 3.6 x 10-6 M for Mg2+-ATPase. One millimolar EDTA prevented the enzyme inhibition induced by CuSO4, but also reversed the inhibited activity, in a concentration-dependent manner, following exposure of the enzymes to the metal ion, by lowering 'free' Cu2+ concentration. Kinetic analysis showed that CuSO4 inhibits both the Na+/K+-ATPase and Mg2+- ATPase, by reducing their maximum enzymatic velocities (V(max)), rather than apparent affinity for substrate MgATP2- (K0.5), implying the noncompetitive nature of enzyme inhibition induced by the metal. The kinetic analysis also confirmed two distinct Mg2+-ATPase subtypes activated in the presence of low and high MgATP2- concentrations. K0.5 and V(max) were calculated using a computer-based program. The results of calculation showed that MgATP2- concentration in the kinetic experiments exceeded three times the apparent K0.5 value for the enzyme activation.