Prevention and recovery of CuSO4-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 MgATP(2-) 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 MgATP(2-) (K-0.5), 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 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. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.