Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na+/K+-ATPase and Mg2+-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu2+, Zn2+, Fe2+ and Co2+) and heavy metals (Hg(2+)and Pb2+) ions were studied. All investigated metals produced a larger maximum inhibition of Na+/K+-ATPase than Mg2+-ATPase activity. The free concentrations of the key species (inhibitor, MgATp(2-), MeATp(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu2+/Fe2+ or Hg2+/Pb(2+)caused additive inhibition, while Cu2+/Zn2+ or Fe2+/Zn2+ inhibited Na+/K+-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu2+/Fe2+ or Cu2+/Zn2+ inhibited Mg2+-ATPase activity synergistically, while Hg2+/Pb2+ or Fe2+/Zn2+ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na+/K+-ATPase activity by reducing the maximum velocities (V-max) rather than the apparent affinity (K-m) for substrate MgATp(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg2+-ATPase activity by Zn2+, Fe2+ and Co2+ as well as kinetic analysis indicated two distinct Mg2+-ATPase subtypes activated in the presence of low and high MgATp2- concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na+/K+-ATPase with various potencies. Furthermore, these ligands also reversed Na+/K+-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg2+-induced inhibition was not obtained

Detection of toxic metal ions in water based on Na+/K+-ATPase activity measurement

The possibility of using enzyme Na+/K+-ATPase as a biological component of sensing system for detection of toxic metal ions (Hg2+, Pb2+ and Cd2+) in water samples has been studied. The method is based on the spectrophotometric determination of inorganic orthophosphate (P) that serves as a measure of the enzymatic activity in the presence of considered metal ions. The concentration of Pi, liberated by enzyme catalysed hydrolysis of adenosinetriphosphate (ATP), was followed spectrophotometrically, by single exposure to analytes or in the mixture. Hg2+, Pb2+ and Cd2+ induced the dose-dependent inhibition of Na+/K+-ATPase activity in enzymatic mixture. Sigmoid shaped inhibition curves were obtained in all cases. Half-maximum inhibitory activities (IC50) were determined by fitting the experimental data by sigmoid function. Considering Na+/K+-ATPase high sensibility to toxic metal ions, the development of simple and quick method for preliminary detection of water contamination by metals based on enzymatic activity measurement was suggested.9th International Conference on Environmental Science and Technology, Sep 01-03, 2005, Rhodes Isl, Greec

Evaluation of photochemical degradation of digoxin by Na,K-ATPase assay

A simple Na,K-ATPase assay is described as a suitable method for testing of digoxin photodegradation. The exposure of Na,K-ATPase to the photodegraded samples exhibited reduced inhibition of the enzyme, compared to the unirradiated samples containing equal initial concentrations of drug. The degree of inhibition was dependent on the irradiation time. The concentrations of digoxin in irradiated samples were evaluated by HPLC analysis. Excellent agreement of the results obtained by both methods was observed. The investigation of the influence of irradiated samples on Na,K-ATPase inhibition revealed no side products acting as Na,K-ATPase inhibitors. The cytokinesis block micronucleus test (CBMN) was applied in order to investigate the cytotoxicity of the possible degradation products after exposure to UV irradiation. The results confirmed that the photochemical treatment did not induce the cytotoxic side products. Zero order kinetics, which was observed for digoxin photodegradation and the associated reaction mechanism are also discussed. (c) 2005 Elsevier B.V. All rights reserved

Detection of toxic metal ions in water based on Na+/K+-ATPase activity measurement

The possibility of using enzyme Na+/K+-ATPase as a biological component of sensing system for detection of toxic metal ions (Hg2+, Pb2+ and Cd2+) in water samples has been studied. The method is based on the spectrophotometric determination of inorganic orthophosphate (P) that serves as a measure of the enzymatic activity in the presence of considered metal ions. The concentration of Pi, liberated by enzyme catalysed hydrolysis of adenosinetriphosphate (ATP), was followed spectrophotometrically, by single exposure to analytes or in the mixture. Hg2+, Pb2+ and Cd2+ induced the dose-dependent inhibition of Na+/K+-ATPase activity in enzymatic mixture. Sigmoid shaped inhibition curves were obtained in all cases. Half-maximum inhibitory activities (IC50) were determined by fitting the experimental data by sigmoid function. Considering Na+/K+-ATPase high sensibility to toxic metal ions, the development of simple and quick method for preliminary detection of water contamination by metals based on enzymatic activity measurement was suggested.9th International Conference on Environmental Science and Technology, Sep 01-03, 2005, Rhodes Isl, Greec

ATPases as multi-response sensing system for various organic and inorganic analytes

The possibility of using synaptic plasma membrane (SPM) enzymes Na+/K+-ATPase and Mg2+-ATPase, isolated from rat brain, as a biological component of multi-response sensing system for detection of different compounds (alkaline and heavy metal salts, organic compounds) was studied. The method is based on the spectrophotometric determination of inorganic ortho-phosphate (P-i) that serves as a measure of the enzymatic activity in the presence of various analytes. The concentration of P-i, liberated by enzyme catalysed hydrolysis of adenosinetriphosphate (ATP), was followed spectrophotometrically, by single exposure to analytes or in the mixture. P-i was dose dependent on the analyte concentration. Alkaline elements (Na, K, Mg), heavy metals (Pb, Cd, Hg, Cu, Fe, Co, Zn), toxic organic compounds (pyridine, urea, chlorpyrifos), and some drugs (digoxin, gitoxin) showed diverse effects, inducing the inhibition or stimulation of the enzymes activity. Development of simple test method for simultaneous detection of the investigated analytes based on the variation of medium assay composition was discussed

Effects of Some Organic and Inorganic Compounds on Atpase Activity

The influence of some organic compounds (pyridine, urea, chlorpyrifos, permethrin) and metal ions (Pb2+, Cd2-, Hg2+, Cu2+, Fe2+ and Zn2+) on Na+/K+-ATPase and Mg2+-ATpase activity isolated from rat brain synaptic plasma membranes (SPM) was investigated in order to develop a simple semi-quantitative and qualitative test method for selective detection of these analyites in aqueous solutions. The method is based on the spectrophotometric determination of inorganic ortho-phosphate (P-i) that serves as a measure of the enzymatic activity in the presence of various analytes. The concentration of P-p liberated by enzyme-catalysed hydrolysis of adenosinetriphosphate (ATP), by exposure to analytes was dose-dependent on the analyte concentration. Heavy metals (Pb, Cd, Hg, Cu, Fe and Zn), toxic organic compounds (pyridine, urea) and some pesticides (ehlorpyrifos, permethrin) showed diverse effects, inducing the inhibition or stimulation of the enzyme activity. Development of simple test method for simultaneous detection of the investigated analytes based on the variation of medium assay composition was discussed

The influence of malathion and its decomposition products on free and immobilized acetylcholinesterase

The influence of malathion and its four main degradation products found in irradiated solutions (malaoxon, isomalathion, diethyl maleate and O,O-dimethyl phosphate) on acetylcholinesterase (AChE) of free and immobilized bovine erythrocytes was investigated. The concentration-dependent responses to malathion and related organophosphates, malaoxon and isomalathion, of both AChE bioassays used were obtained. The IC (50) values for free and immobilized AChE (3.7 +/- 0.2) x 10(-4) M/(1.6 +/- 0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6) M, and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M were obtained in the presence of malathion, malaoxon and isomalathion, respectively. However, diethyl maleate inhibited AChE activity at concentrations GT = 10 mM, while O,O-dimethyl phosphate did not noticeably affect enzyme activity at all investigated concentrations. The relation between the structure of the compounds and their ability to inhibit enzyme activity was discussed

Effects of digoxin and gitoxin on the enzymatic activity and kinetic parameters of Na+/K+-ATPase

Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1 LT n LT 3; suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (V-max) and K-m, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration

Inhibition of AChE by single and simultaneous exposure to malathion and its degradation products

In vitro inhibition of bovine erythrocytes acetylcholinesterase (AchE) by separate and simultaneous exposure to organophosphorous insecticide malathion and the transformation products, which are generally formed during the storage or natural as well as photochemical degradation pathways of malathion, was investigated. The increasing concentration of malathion, its oxidation product - malaoxon and isomerisation product - isomalathion inhibited AChE activity in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC50 values): (3.2 +/- 0.1) x 10(-5) mol/l, (4.7 +/- 0.8) x 10(-7) mol/l and (6.0 +/- 0.5) x 10(-7) mol/l were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl phosphorodithioic ester (OOS(S)) and O,O-dimethyl thiophosphate did not noticeably affect the enzyme activity at all investigated concentrations, while diethyl maleate inhibited the AChE activity at concentrations GT 10 mmol/l. By simultaneous exposure of the enzyme to malaoxon and isomalathion in various concentration combinations the additive effect was achieved by low concentration of inhibitors, while the antagonistic effect was obtained at high concentration ( GT = 3 x 10-7 mol/l) of inhibitors. Inhibitory power of irradiated samples of 1 x 10(-5) mol/l malathion can be attributed to the formation of malaoxon and isomalathion, organophosphates about 100 times more toxic than their parent compound, while the presence of non-inhibiting degradation product OOS(S) did not affect the inhibitor efficiency of inhibiting malathion by-products, malaoxon and isomalathion

Time-dependent inhibition of Na+/K+-ATPase induced by single and simultaneous exposure to lead and cadmium

Time-dependent interactions of Na+/K+-ATPase, isolated from rat brain synaptic plasma membranes (SPMs), with Cd2+ and Pb2+ ions in a single exposure and in a mixture were investigated in vitro. The interference of the enzyme with these metal ions was studied as a function of different protein concentrations and exposure time. The aim of the work was to investigate the possibility of selective recognition of Cd2+ and Pb2+ ions in a mixture, on the basis of the different rates of their protein-ligand interactions. Decreasing protein concentration increased the sensitivity of Na+/K+-ATPase toward both metals. The selectivity in protein-ligand interactions was obtained by variation of preincubation time (incubation before starting the enzymatic reaction).8th International Conference on Fundamental and Applied Aspects of Physical Chemistry, Sep 26-29, 2006, Belgrade, Serbi