Prevention and recovery of CuSO 4-induced inhibition of Na +/K +-ATPase and Mg 2+-ATPase in rat brain synaptosomes by EDTA

Abstract

Enzymatic activities of Na +/K +-ATPase and Mg 2+-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 CuSO 4-induced inhibition. The influence of experimentally added CuSO 4 and EDTA on MgATP 2− complex and ‘free’ Cu 2+ concentrations in the reaction mixture was calculated and discussed. CuSO 4 induced dose-dependent inhibition of both enzymes in the absence and presence of 1 mM EDTA. In the absence of EDTA, the IC 50 values of Cu 2+, as calculated from the experimental curves, were 5.9×10 −7 M for Na +/K +-ATPase and 3.6×10 −6 M for Mg 2+-ATPase. One millimolar EDTA prevented the enzyme inhibition induced by CuSO 4, but also reversed the inhibited activity, in a concentration-dependent manner, following exposure of the enzymes to the metal ion, by lowering ‘free’ Cu 2+ concentration. Kinetic analysis showed that CuSO 4 inhibits both the Na +/K +-ATPase and Mg 2+-ATPase, by reducing their maximum enzymatic velocities ( V max), rather than apparent affinity for substrate MgATP 2− ( K 0.5), implying the noncompetitive nature of enzyme inhibition induced by the metal. The kinetic analysis also confirmed two distinct Mg 2+-ATPase subtypes activated in the presence of low and high MgATP 2− concentrations. K 0.5 and V max were calculated using a computer-based program. The results of calculation showed that MgATP 2− concentration in the kinetic experiments exceeded three times the apparent K 0.5 value for the enzyme activation.