Prevention and recovery of (μ 3-diethylentriamino)-chloro-palladium(II)-chloride induced inhibition of Na/K-ATPase by SH containing ligands – l-cysteine and glutathione

Abstract

The effect of (μ 3-diethylentriamino)-chloro-palladium(II)-chloride ([PdCl(dien)]Cl) on the activity of Na/K-ATPase from porcine cerebral cortex was studied in vitro, in the absence and presence of –SH containing ligands l-cysteine and glutathione (GSH). The aim of the study was to elucidate the mechanism of [PdCl(dien)] + induced inhibition of the enzyme activity and to examine the ability of thiols to prevent and recover the inhibition. The coordinative interaction between [PdCl(dien)] + and enzyme was verified by UV and 1H NMR spectra. The semblance in the changes in absorption spectra of [PdCl(dien)] + in the presence of Na/K-ATPase and thiols ( l-cysteine and GSH) suggested that the complex ion interacts with enzymatic sulfhydryl groups. [PdCl(dien)] + inhibited the enzyme activity in a dose-dependent manner. The Hill analysis of the inhibition curve yielded the half-maximum inhibitory activity value, IC 50 = 1.21 × 10 −4 M, and Hill coefficient, n = 0.7, suggesting the negative cooperation for binding of [PdCl(dien)] + to the enzyme. Dependence of the initial reaction rate on the concentration of MgATP 2− exhibited typical Michelis–Menten kinetics in the absence and presence of the inhibitor. Kinetic analysis showed that [PdCl(dien)] + inhibited Na/K-ATPase by reducing the maximum reaction rate ( V max), rather than changing the affinity to the substrate ( K m). Kinetic parameters derived using Lineweaver–Burk transformation of experimental data indicated the non-competitive nature of Na/K-ATPase inhibition. The inhibitory constant, K i = 1.05 × 10 −4 M, was determined from secondary replot of Lineweaver–Burk graph, and correlated with stability constants of [Pd(dien)(thiol)] complexes. 1 × 10 −3 M l-cysteine or GSH prevented the enzyme inhibition induced by Pd(II) complex cation when present below 1 × 10 −4 M. The both thiols completely reversed the inhibited activity in the concentration dependent manner, due to the complex formation with [PdCl(dien)] +.