Krinulović Katarina, Bugarcić Zivadin, Vrvić Miroslav, Krstić Danijela, Vasić Vesna
Vinca Institute of Nuclear Sciences, Department of Physical Chemistry, P.O. Box 522, 11001 Belgrade, Serbia and Montenegro.
Toxicol In Vitro. 2006 Dec;20(8):1292-9. doi: 10.1016/j.tiv.2006.03.002. Epub 2006 Mar 8.
The effect of (mu(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 (1)H 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 x 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 x 10(-4)M, was determined from secondary replot of Lineweaver-Burk graph, and correlated with stability constants of [Pd(dien)(thiol)] complexes. 1 x 10(-3)M L-cysteine or GSH prevented the enzyme inhibition induced by Pd(II) complex cation when present below 1 x 10(-4)M. The both thiols completely reversed the inhibited activity in the concentration dependent manner, due to the complex formation with PdCl(dien).
研究了（μ(3)-二亚乙基三胺）-氯-钯（II）-氯化物（[PdCl(dien)]Cl）在体外对猪脑皮层Na/K-ATP酶活性的影响，实验分别在不存在和存在含巯基配体L-半胱氨酸和谷胱甘肽（GSH）的条件下进行。本研究的目的是阐明[PdCl(dien)]⁺诱导酶活性抑制的机制，并考察硫醇预防和恢复这种抑制的能力。通过紫外光谱和¹H NMR光谱验证了[PdCl(dien)]⁺与酶之间的配位相互作用。在存在Na/K-ATP酶和硫醇（L-半胱氨酸和GSH）的情况下，[PdCl(dien)]⁺吸收光谱变化的相似性表明该络合离子与酶的巯基相互作用。[PdCl(dien)]⁺以剂量依赖性方式抑制酶活性。对抑制曲线进行希尔分析得到半数最大抑制活性值IC₅₀ = 1.21×10⁻⁴M，希尔系数n = 0.7，表明[PdCl(dien)]⁺与酶结合存在负协同效应。在不存在和存在抑制剂的情况下，初始反应速率对MgATP²⁻浓度的依赖性均表现出典型的米氏动力学。动力学分析表明，[PdCl(dien)]⁺通过降低最大反应速率（Vmax）而非改变对底物的亲和力（Km）来抑制Na/K-ATP酶。利用实验数据的Lineweaver-Burk变换得到的动力学参数表明Na/K-ATP酶抑制作用的非竞争性本质。从Lineweaver-Burk图的二次重图确定抑制常数Kᵢ = 1.05×10⁻⁴M，并与[Pd(dien)(硫醇)]络合物的稳定常数相关。当浓度低于1×10⁻⁴M时，1×10⁻³M的L-半胱氨酸或GSH可预防由钯（II）络合阳离子诱导的酶抑制。由于与[PdCl(dien)]⁺形成络合物，两种硫醇均以浓度依赖性方式完全逆转被抑制的活性。
