Prevention and recovery of CuSO4-induced inhibition of Na+/K+-ATPase and Mg2+-ATPase in rat brain synaptosomes by EDTA

Enzymatic activities of Na+/K+-ATPase and Mg2+-ATPase from rat brain synapt ic plasma membrane were studied in the absence and presence of EDTA. The ai m of the study was to examine the ability of this strong chelator to preven t and recover the CuSO4-induced inhibition. The influence of experimentally added CuSO4 and EDTA on MgATP(2-) complex and 'free' Cu2+ concentrations i n the reaction mixture was calculated and discussed. CuSO4 induced dose-dep endent 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 ex perimental 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 indu ced by CuSO4, but also reversed the inhibited activity, in a concentration- dependent manner, following exposure of the enzymes to the metal ion, by lo wering 'free' Cu2+ concentration. Kinetic analysis showed that CuSO4, inhib its both the Na+/K+-ATPase and Mg2+-ATPase, by reducing their maximum enzym atic velocities (V-max), rather than apparent affinity for substrate MgATP( 2-) (K-0.5), implying the noncompetitive nature of enzyme inhibition induce d by the metal. The kinetic analysis also confirmed two distinct Mg2+-ATPas e subtypes activated in the presence of low and high MgATP(2-) concentratio ns. K-0.5 and V-max were calculated using a computer-based program. The res ults of calculation showed that MgATP(2-) concentration in the kinetic expe riments exceeded three times the apparent K-0.5 value for the enzyme activa tion. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.