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

Abstract

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 (Hg²⁺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⁺/Pb²⁺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 MgATP^{2 −} 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.

Keywords::

• Transition and heavy metals
• Na⁺/K⁺-ATPase
• Mg²⁺-ATPase
• kinetics
• inhibition

Introduction

Na⁺/K⁺-ATPase and Mg²⁺-ATPase are membrane enzymes ubiquitous in animal cells that involve adenosine triphosphate (ATP) as a substrate for their functioning 1-3. The ouabain-sensitive Na⁺/K⁺-ATPase, or sodium pump, is a membrane bound protein that establishes and maintains the high internal K⁺ and low internal Na⁺ concentrations, characteristic of most animal cells. The ouabain-insensitive Mg²⁺-ATPase is much less well characterized, but apparently consists of at least two forms with different molecular weights [4] and sensitivity to metal ions [5,6].

Hydrolysis of ATP catalysed by these ATPases supplies the energy required for many biochemical processes [7] and depends sensitively on pH, temperature, presence of metal ions, such as Na⁺, K⁺ and Mg²⁺ as well as presence of some modulators. It was reported that highly toxic heavy metals induced inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities in a concentration-dependent manner 8-11. The metals of the first transition series (Fe, Co, Cu, Zn) are apparently necessary to some physiological processes, but as previously reported, the higher concentrations alter plasma membrane functioning in a variety of tissues, both in vitro and in vivo 12-14. Inhibitory effects of Cu²⁺, Zn²⁺ and Fe²⁺ on bovine cerebral cortex Na⁺/K⁺-ATPase activity have been obtained and the extent of inhibition seemed to depend on the presence of chelators [15].

Recently, we investigated the in vitro influence of some metal ions (Cu²⁺, Co²⁺, Hg²⁺, Cd²⁺) on Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity using rat synaptic plasma membranes (SPM) as a model system [5,6,8,9,16]. The present paper continues our study and investigates the effects of some first transition series elements (Cu²⁺, Zn²⁺, Fe²⁺, Co²⁺) and heavy metals (Hg²⁺ and Pb²⁺) on the activity of SPM Na⁺/K⁺-ATPase and Mg²⁺-ATPase. On the one hand, these effects were induced by separate, and on the other, by simultaneous exposures to investigated metals. It has been shown that some of the investigated ions (Cu²⁺, Zn²⁺ and Fe²⁺) form complex entities with ATP. Therefore, our aim was to elucidate whether the inhibition was caused by a decrease in substrate (MgATP^{2 −} ) concentration or was due to interaction with the enzyme. Consequently we followed the influence of the investigated metals on MgATP^{2 −} concentration in the enzyme medium. We also investigated the abilities of − SH containing ligands (gluthathione and l-cysteine) and EDTA to prevent and recover the metal ion – induced inhibition since chelators are able to protect against some toxic effects of metal ions [17]. In addition, extensive kinetic studies were undertaken to determine the nature of the enzyme inhibition by the investigated metals.

Material and methods

Chemicals

All chemicals for the medium assay were commercially available from Sigma (St. Louis, MO, USA) and were of reagent grade. The metal ion salts: mercury (II) chloride, lead (II) chloride, cobalt (II) chloride, zinc (II) chloride, cooper (II) chloride, iron (II) chloride, NaCl, KCl, stannous chloride, ammonium molybdate, were from Merck (Darmstadt, Germany). All solutions were prepared using de-ionized water.

Animals

Adult male Wistar rats three months, 330–400 g, were housed under standard laboratory conditions in the animal house of the Vinča Institute of Nuclear Sciences (Beograd, Serbia & Montenegro). Animals were kept under controlled illumination (lights on: 5:00 a.m.–5:00 p.m.) and temperature (23 ± 2°C), and had free access to food and water. The “Guiding Principles for the Care and Use of Animals” based upon Helsinki Declaration (1964) and “Protocol of the “Vinča” Institute on care and treatment of laboratory animals” were strictly followed.

Synaptic plasma membrane (SPM) preparation

SPM were isolated from the whole rat brain. After decapitation with a guillotine (Harvard Apparatus), brains were rapidly excised and pooled (6/pool) for immediate preparation of synaptic plasma membranes. The SPM were isolated according to the method of Cohen et al. [18], as modified by Towle and Sze [19]. The mitochondrial contamination and protein content were determined according to the standard procedure [16]. SPM were stored at − 70^oC until used.

ATPase assay

The standard assay medium for investigation of ATPases activity contained (in mM) 50 Tris–HCl, pH 7.4; 100 NaCl; 20 KCl; 5 MgCl₂; 2 ATP; 25 μg SPM proteins. After preincubation for 10 min at 37°C in the absence (control) or in the presence of investigated metals, the reaction was initiated by addition of ATP and stopped after 10 min by adding 22 μl ice cold HClO₄ and immediate cooling on ice. The released inorganic orthophosphate (Pi) liberated from the hydrolysis of ATP was determined by a modified spectrophotometric method [6]. The spectrophotometric measurements were performed on a Beckman 5260 UV VIS spectrophotometer. The activity obtained in the presence of Mg²⁺ alone was attributed to Mg²⁺-ATPase activity. Na⁺/K⁺ -ATPase activity was calculated by subtracting the Mg²⁺-ATPase activity from the total ATPase activity in the presence of Na⁺, K⁺ and Mg²⁺ ions. The results are expressed as mean % enzyme activity compared to the corresponding control value ± S.E.M. of at least three independent experiments done in triplicate.

Since some of the investigated metal ions form complexes with ATP, their presence required the monitoring of the concentration of MgATP^{2 −} in the reaction mixture which is the actual substrate for both ATPases. The following reactions in the medium assay were taken into consideration for calculation the concentration of MgATP^{2 −} , MeATP^{2 −} and free, uncomplexed metal ion concentrations:

Free metal ion, MeATP^2– and MgATP^2– concentrations were calculated according to the well-known Storer and Cornish-Bowden method [20] taking into account all the equilibrium reactions involving Mg²⁺, investigated metal ions and ATP. The stability and equilibrium constants were taken from references [21,22].

The effect of EDTA and − SH containing ligands (l-cysteine and glutathione) on the prevention of the metal ion induced inhibition was measured under the same conditions as described above, with the ligand added to the medium assay before the exposure to metal ions. The recovery of inhibited enzyme activity by investigated ligand was examined by adding ligand in the experimental tube, after 10 min of preincubation in the presence of metal ion at a concentration which induced complete enzyme inhibition. The control tubes contained the corresponding concentration of chelators without metal ions.

Kinetic analysis

Kinetic analysis was carried out according to a slightly modified method of Phylips [23], by following the initial velocity of the enzymatic reaction in the presence of inhibitor (concentration near IC₅₀) and rising concentrations of ATP (0.25–5.5 mM), while maintaining the concentrations of other ions (Mg²⁺, Na⁺, K⁺) constant. The data were analyzed by the softwere package developed [6] and the results were recalculated using EZ FIT [24].

Results

Effects of metal ions on SPM ATPase activities and substrate concentrations

The separate influence of metal ions on Na⁺/K⁺- ATPase and Mg²⁺- ATPase activity was investigated in the concentration range 1 × 10^–8–1 × 10^–2 M. The increasing concentrations of metal ions induced inhibition of enzymatic activity in a concentration—dependent manner. Sigmoidal inhibition curves for both enzymes were obtained. The half-maximum inhibitory concentrations (IC₅₀) of the investigated ions for both enzymes were determined by Hill analysis of the experimental results and are summarized in Table I. In addition, since it is generally believed that the ionic form of metal ions is responsible for protein interactions, corresponding IC₅₀ values of the “uncomplexed”,”free” form of metals that form complexes with ATP, were calculated and are presented in Table I. It is clearly apparent that Na⁺/K⁺ -ATPase is more sensitive to all the investigated metals than Mg²⁺- ATPase. It is interesting to note that transition metals (except Cu²⁺) do not inhibit Mg²⁺-ATPase completely, even when present in concentrations above 1 × 10^–3 M. The inhibition of Mg²⁺-ATPase activity asymptotically approaches 57% for Zn²⁺, 80% for Fe²⁺ and 78% for Co²⁺ in contrast to 100% for Na⁺/K⁺-ATPase (results not shown).

Table I.  Experimental and recalculated “free” IC₅₀ values (μM).

	IC50 values (μM)
	Na^+/K^+-ATPase		Mg^2+-ATPase
Metal ion	Exp.	Calc.	Exp.	Calc.
Fe^2+	34	–	170	–
Co^2+^a	168	75	262	136
Cu^2+^b	7.1	0.6	42	33
Zn^2+	22	13	108	53
Hg^2+	0.7	–	3.6	–
Pb^2+	7.5	–	13.6	–

IC₅₀ values; ^aref. 5; ^bref. 6.

The dependence of MgATP^2– concentration on metal concentration in the assay medium assay at pH 7.4 is presented in Figure 1. The results show that at a concentration level below 2 × 10^–4 M, metal ions do not have a significant effect on substrate concentration in the enzyme mixture since under the experimental conditions, the concentration of MgATP^2– was in excess. By increasing metal ion concentration above 2 × 10^–4 M, MgATP^2– concentration significantly decreased.

Figure 1 The dependence of MgATP^2– concentration in the reaction mixture containing 2 mM ATP and 5 mM MgCl₂ on the experimental Me²⁺ concentration: (▾) Cu²⁺; (▴) Co²⁺; (▪) Zn²⁺; (•) Fe²⁺, Hg²⁺, Pb²⁺.

Inhibition of SPM ATPases by simultaneous exposure to metal ions

The effect of simultaneous exposure to combinations of metal ions on the activity of Na⁺/K⁺-ATPase and Mg²⁺-ATPase was studied by using the mixtures of Cu/Fe, Cu/Zn, Fe/Zn, and Pb/Hg. The concentrations of metals were 5 × 10^–7–2 × 10^–5 M, i.e. limited to producing the inhibition in the concentration range lower than the IC₅₀ values. The inhibition obtained by exposure to several combinations was compared to the inhibition in the presence of each single metal. The mean values of experimentally determined and mathematically added values are presented in Table II. The mathematically determined means were calculated as the sum of metal ion-induced inhibitions measured separately. Analysis of variance (One way ANOVA) was used to compare the mathematical sum of inhibitions caused by separate exposure to metal ions with inhibitions induced by exposure to two metals in combination (simultaneously). When a significant F value (P < 0.05) was obtained, the post hoc test Bonferoni was used to determine the differences.

Table II.  Inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity by simultaneous exposure to metal ions in mixtures.

	% Inhibition
	Na^+/ K^+-ATPase		Mg^2+-ATPase
Concentration (μM)	Experimental	Calculated	Experimental	Calculated
0.5 Cu +2 Fe	16.6 ± 2.5	17.2 ± 1.6	7.2 ± 1.3	5.2 ± 1.6
0.5 Cu +20 Fe	26.3 ± 2.8	28.6 ± 3.2	22.5 ± 2.1	3.2 ± 0.8
1 Cu +2 Fe	18.9 ± 1.1	20.4 ± 1.2	7.3 ± 0.9	5.0 ± 0.5
1 Cu +20 Fe	30.7 ± 2.2	32.8 ± 1.3	22.5 ± 2.9	6.4 ± 1.7
0.5 Cu +2 Zn	41.2 ± 2.5	11.6 ± 3.1	12.4 ± 2.4	4.1 ± 1.1
0.5Cu +20 Zn	65.9 ± 4.2	46.2 ± 2.1	17.8 ± 2.4	4.3 ± 0.9
1 Cu +2 Zn	68.4 ± 5.1	13.3 ± 2.3	13.9 ± 3.6	5.2 ± 1.5
1 Cu +20 Zn	100 ± 6.5	49.9 ± 3.6	18.7 ± 1.9	5.5 ± 1.0
2 Fe +2 Zn	20.8 ± 2.2	10.8 ± 2.1	1.2 ± 0.3	3.0 ± 0.2
2 Fe +20 Zn	85.1 ± 3.6	50.4 ± 1.1	3.9 ± 0.7	7.3 ± 1.3
20 Fe +2 Zn	65.1 ± 1.3	40.3 ± 1.4	1.6 ± 0.4	21.1 ± 2.1
20 Fe +20 Zn	100 ± 2.1	61.9 ± 3.3	3.6 ± 0.5	22.3 ± 1.9
1 Pb +0.5 Hg	42.2 ± 2.0	44.5 ± 1.5	14.2 ± 0.3	23.0 ± 1.3
5 Pb +0.5 Hg	75.5 ± 3.3	73.1 ± 1.2	38.7 ± 0.7	53.1 ± 3.3

A synergistic effect was defined as a statistically significant (P < 0.05) difference between inhibitions caused by simultaneous exposure and mathematically calculated mean values of enzyme inhibition for a pair of metals, where the former is larger than the latter. An antagonistic effect was defined as above, except that the inhibition caused by simultaneous exposure is less than the mathematically calculated mean value.

Cu²⁺ and Fe²⁺ together caused an additive inhibition, while simultaneous exposure to Cu²⁺ and Zn²⁺, or Fe²⁺ and Zn²⁺ induced synergistic inhibition of SPM Na⁺/K⁺- ATPase activity at all investigated concentrations. Simultaneous exposure of Mg²⁺-ATPase to Cu²⁺ and Fe²⁺ or Cu²⁺ and Zn²⁺ ions caused synergistic inhibition, while Fe²⁺ and Zn²⁺ together inhibited the enzyme antagonistically. Exposure to the Pb²⁺/Hg²⁺ combination induced an additive inhibition of Na⁺/K⁺- ATPase activity as well as antagonistic inhibition of Mg²⁺-ATPase activity at all concentrations examined.

Effects of chelators on the prevention and recovery of inhibited Na⁺/K⁺-ATPase activity

The inhibition of Na⁺/K⁺-ATPase induced by metal ions was investigated in the presence of 1 mM EDTA, 10 mM l-cysteine and 10 mM GSH. The Zn²⁺-induced inhibition curves of Na⁺/K⁺-ATPase activity in the presence and absence of chelators are shown in Figure 2, and similar results were obtained for the other metals of the first transition series. It is obvious that the presence of EDTA in the reaction mixture at a 1 mM concentration prevents enzyme inhibition at a metal concentration below 1 mM, since 10 mM l-cysteine and 10 mM GSH induce the same effect. Moreover, in the presence of 1 mM EDTA, or 10 mM sulphur-containing chelators, the effect on the enzyme activity of these investigated metals in the reaction mixture at a total concentration below 1 × 10^–4 M was negligible.

Figure 2 Inhibition of Na⁺/K⁺-ATPase by Zn²⁺ in the absence (○) and presence of: 10 mM l-Cysteine (▪); 10 mM glutathione (▴) and 1 mM EDTA (•).

The Hg²⁺-induced inhibition curves of Na⁺/K⁺-ATPase activity in the presence and absence of chelators are shown in Figure 3. It could be noted that the most protective effect on Na⁺/K⁺-ATPase activity was achieved in the presence of 10 mM GSH in the reaction mixture, while the ability of EDTA to prevent the Hg²⁺-induced inhibition was the least. In the presence of 10 mM GSH, the IC₅₀ value of HgCl₂ was almost five orders of magnitude higher than the IC₅₀ value in the absence of any chelator, while the presence of 10 mM l-cysteine caused a four orders of magnitude increase in the IC₅₀ value for HgCl₂.

Figure 3 Inhibition of Na⁺/K⁺-ATPase by Hg²⁺ in the absence (○) and presence of: 1 mM EDTA (•); 10 mM l-cysteine (▪) and 10 mM glutathione (▴).

Since it was demonstrated that EDTA, l-cysteine and GSH prevent the metal ion-induced inhibition of Na⁺/K⁺-ATPase, the recovery of the inhibited enzyme activities was investigated by varying the chelator concentration from 1 × 10^–6–1 × 10^–1 M. The effect of chelators was investigated in the presence 1 × 10^–4 M of metal ions, given the fact that at this concentration, investigated metals induced complete inhibition of Na⁺/K⁺-ATPase. The results presented in Figure 4 show that the chelators had a dose-dependent recovery effect on Na⁺/K⁺-ATPase activity exposed to all metal ions that belong to the first transition series. On the contrary, recovery of Hg²⁺-induced inhibition was not achieved, even with a chelator's concentrations above 0.01 M.

Figure 4 Effects of chelators: EDTA (•); l-cysteine (▪) and glutathione (▴) on the recovery of Na⁺/K⁺-ATPase activity in the presence of 1 × 10^–4 M ZnCl₂.

Kinetic analysis

In order to evaluate the nature of metal ion-induced Na⁺/K⁺-ATPase and Mg²⁺-ATPase inhibition, kinetic analysis in the presence and absence of inhibitors was performed. The influence of metal ions (concentrations near IC₅₀ values) on catalytic activity was investigated by following the initial velocities vs. the concentration of ATP (from 0.25–5.50 mM). Dependence of the reaction rate vs. MgATP^{2 −} concentration for Na⁺/ K⁺-ATPase in the presence and absence of metal ions exibited typical Michaelis–Menten kinetics in all cases. The kinetic parameters (K_m, V_max) were estimated from Eadie–Hofstee transformation of the data and are summarized in Table III. It is obvious that all metal ions that belong to the first transition series inhibited enzyme activity noncompetitively by decreasing its V_max value, without changing significantly its apparent affinity for ATP. It is worthy to note that the saturating levels of MgATP^2– were always present in the assay medium assay at an IC₅₀ of metal ions.

Table III.  Kinetic analysis of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity in the absence (control) and presence of metal ions.

			Mg^2+-ATPase
	Na^+/K^+-ATPase		High affinity		Low affinity
	Km (mM)	Vmax (μM Pi/mg/h)	Km (mM)	Vmax (μMPi/mg/h)	S0.5	Vmax (μM Pi/mg/h)
Control	0.69 ± 0.05	46.10 ± 2.20	0.32 ± 0.05	68.18 ± 1.20	3.79 ± 0.06	19.80 ± 0.90
Fe^2+	0.72 ± 0.09	25.80 ± 0.03	0.32 ± 0.04	57.80 ± 0.05	3.70 ± 0.04	19.80 ± 0.20
Co^2+	0.75 ± 0.09	33.20 ± 0.12	0.31 ± 0.03	55.20 ± 0.09	3.72 ± 0.06	18.90 ± 0.90
Cu^2+	0.74 ± 0.05	31.80 ± 0.80	0.33 ± 0.02	54.00 ± 1.20	3.64 ± 0.10	15.60 ± 1.00
Zn^2+	0.70 ± 0.09	19.60 ± 0.20	0.33 ± 0.01	56.70 ± 0.30	3.73 ± 0.03	19.73 ± 0.90

Experimental concentration of metal ions: Zn²⁺ = 1.5 × 10^–5 M; Cu²⁺ = 7.5 × 10^–6 M; Fe²⁺ = 3.4 × 10^–5 M; Co²⁺ = 1 × 10^–4 M.

Mg²⁺-ATPase activity exhibited biphasic dependence on increasing MgATP^2– concentration in the absence and presence of all investigated metal ions. The Mg²⁺-ATPase activity as a function of MgATP^2– concentration, in the presence and absence of ZnCl₂ is presented in Figure 5(a). The obtained functions are not purely of the Michaelis–Menten type, and could be approximated by two overlapping curves separated by a plateau. The results indicate that the enzyme activity at each ATP concentration represent the sum of the activity of two distinct Mg²⁺-ATPase subtypes i.e. the plot of the total activity represents the line for “two enzymes acting on one substrate”. The first one, active at a substrate concentration below 1 mM, is denoted as “high affinity”, while the second, active at MgATP^2– concentration above 2 mM, is denoted as “low affinity” Mg²⁺-ATPase. Analysis of the data by a PC software package [20] revealed the Michaelis–Menten enzyme kinetics at low and sigmoidal kinetics at high ATP concentrations. The theoretical curves for “high” and “low affinity” Mg²⁺-ATPase subtypes in the presence and absence of Zn²⁺ are presented in Figure 5(b). The kinetic parameters for “high affinity” subtype, in the presence and absence of metal ions, were calculated by the Eadie–Hofstee transformation and are summarized in Table III. These results suggest that Zn²⁺, Fe²⁺, Co²⁺and Cu²⁺ are noncompetitive inhibitors of this enzyme, because they significantly decrease its V_max value without changing significantly its apparent affinity for ATP. The activity of the “low affinity” subtype was reconstructed by subtracting the calculated “high affinity” values at each ATP concentration from the experimentally measured total activity. From the obtained results, initial velocity was recalculated. The iterative procedure was repeated until the best fit of the experimental results was achieved. The kinetic constants of the “low affinity” Mg²⁺-ATPase were calculated by Hill analysis and are presented in Table III. It is notable that the effects of Zn²⁺, Fe²⁺and Co²⁺ on the kinetic parameters of the “low affinity” Mg²⁺-ATPase was negligible.

Figure 5 (a) The dependence of Mg²⁺-ATPase activity on MgATP^2– in the absence (▴) and in the presence (Δ) of 15 μM ZnCl₂. Symbols represent experimental points. (b) The Mg²⁺-ATPase theoretical kinetic curves (Mg²⁺-ATPase activity vs MgATP^{2 −} concentration) of: “high affinity “Mg²⁺-ATPase subtype (○) in the absence of ZnCl₂ (•) and in the presence of ZnCl₂ (○); and “low affinity” Mg²⁺-ATPase subtype (squares) in the absence of ZnCl₂ (▪) and in the presence of ZnCl₂ (▪ or □).

Discussion

Results of our study showed that the examined metal ions, as previously reported [5,6,9,25], inhibited rat brain Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities and had various potencies (Table I). It is reasonable to assume that this potency depends on the nature of the metal ion as well as the nature of the enzyme. Obtained IC₅₀ values of Zn²⁺ and Fe²⁺ for Na⁺/K⁺-ATPase are of the same order of magnitude as the IC₅₀ values reported for bovine cerebral cortex Na⁺/K⁺-ATPase [15]. Experimental IC₅₀ values (Table I) of all investigated ions that form inactive MeATP^2– complex are below 2 × 10^–4 M for both enzymes, while the concentration of substrate, MgATP^2–, remains practically unchanged (Figure 1).These results indicate that inhibition of the enzyme activities was due to the metal ion binding on the enzyme binding sites rather than the influence on the formation of an inactive MeATP^2– complex.

Furthermore, incomplete inhibition of Mg²⁺-ATPase by Zn²⁺, Fe²⁺ and Co²⁺ is in accordance with kinetic analysis that confirms the existence of at least two Mg²⁺-ATPase subtypes. One of them, denoted as “low affinity” Mg²⁺-ATPase, being insensitive towards the inhibition induced by Zn²⁺, Fe²⁺ and Co²⁺ and their presence, had no effect on kinetic parameters. Incomplete inhibition of Mg²⁺-ATPase activity supports the results of earlier studies of rat brain ATPases which suggested that there are two different Mg-ATPases differentiated by their sensitivity to metal ions [25] and ehacrinic acid [4]. Two distinct Mg²⁺-ATPase activities differentiated by their sensitivity to Ca²⁺, La³⁺ and F^-, were isolated from Triton-X-100 solubilized human erythrocyte membranes [26]. On the contrary, as previously reported [6,9], Cu²⁺ and heavy metals (Pb²⁺, Cd²⁺ and Hg²⁺) inhibit Mg²⁺-ATPase activity completely, while significantly changing the kinetic parameters for both Mg²⁺-ATPases. These results are also in accordance with synergistic inhibition caused by simultaneous exposure to Cu²⁺ and Zn²⁺ or Cu²⁺ and Fe²⁺ suggesting different modes of interaction of Cu²⁺ and Zn²⁺ (as well as Cu²⁺ and Fe²⁺) with Mg²⁺-ATPases.

The additive inhibition of SPM Na⁺/ K⁺-ATPase activity obtained by simultaneous exposure to Cu²⁺ and Fe²⁺ (as well as Hg²⁺ and Pb²⁺) suggests that both metal ions are competing for the same inhibitor binding sites and there is an excess of inhibitor binding sites within the concentration ranges examined. Competition between Hg²⁺ and Pb²⁺ is in agreement with the chemical similarities of these ions [21,22], as well as with the previously reported fact that both ions are noncompetitive inhibitors of the investigated enzyme [9]. The results that Cu²⁺ in combination with Zn²⁺, as well as Fe²⁺ in combination with Zn²⁺, inhibit Na⁺/K⁺-ATPase activity synergistically, but additivelly with each other (Cu²⁺/Fe²⁺), suggest that Zn²⁺ binds at different binding sites, which are linked to different but associated mechanisms of inhibition. The Zn²⁺-induced inhibition may include, as suggested earlier [15], Zn²⁺ binding to the Mg²⁺-binding site, which directly affects the phosphorylation step in the enzyme cycle [15], while Cu²⁺- and Fe²⁺-induced inhibition involve interactions with other functional groups on the enzyme. However, Zn²⁺ binding to the Mg²⁺-binding site could not be responsible for Mg²⁺-ATPase inhibition, since this enzyme appears not to belong to the P-type ATPases although synergistic inhibition obtained by simultaneous exposure to Cu²⁺ and Zn²⁺ (as well as Cu²⁺ and Fe²⁺) suggest that these pairs of metals bind to different types of binding site. The antagonistic inhibition of Mg²⁺-ATPase activity with Fe²⁺ and Zn²⁺ at all concentrations examined suggest that the two metals are competing for a limited number of binding sites or the binding of one metal decreases the binding of second through a conformational change in the enzyme.

Our study clearly showed that the presence of 1 mM EDTA, 10 mM l-cysteine and 10 mM GSH prevents enzyme inhibition induced by Zn²⁺, Fe²⁺, Co²⁺ and Cu²⁺. These results are in agreement with the strong chelating effects of the investigated ligands which significantly decrease the ionic form of metals able to interact with enzymes. Strong protective effects of − SH containing-ligands on Hg²⁺-induced inhibition are a sufficient basis for a plausible assumption that Hg²⁺ is a potent reagent for thiol groups. Previous studies have shown that Hg²⁺ inhibition is due to its binding to sulphydryl groups of Na⁺/K⁺-ATPase [27,28], which means that the lowest protective effect of EDTA can be explained by supposing that Hg²⁺ binds more efficiently to the − SH groups of enzyme in comparison to this ligand.

Moreover, the recovery of transition metal-induced inhibition of Na⁺/ K⁺ -ATPase is probably due to competition between the functional groups of protein and ligands for complex formation with these metal ions. These results are consistent with the kinetic analysis suggesting that these ions are reversible, noncompetitive inhibitors of this enzyme. In spite of the fact that the kinetic analysis showed that Hg²⁺—induced inhibition was reversible [9], the attempt to reactivate the enzyme clearly failed. Hence, we may assume that the Hg binding affinity for the enzyme far exceeds its affinity towards either of the investigated chelators.

To summarize, our results confirm that the inhibitory effects of metal ions may be attributed to their ionic forms and that the induced inhibition is likely due to direct interaction between metal ions and enzymes instead of between ions and ATP. Kinetic analysis showed that the nature of Na⁺/ K⁺—ATPase inhibition by the investigated metals is non-competitive. The incomplete inhibition by Zn²⁺, Fe²⁺ and Co²⁺, as well as kinetic analysis indicated two distinct SPM Mg²⁺-ATPase subtypes with different sensitivity to metal ions. The inhibitory effects can be prevented by addition of strong metal—ion chelators, such as EDTA, l-cysteine and GSH. Finally, the effect of these chelators on the recovery of transition metal-induced enzyme inhibition is dose-dependent, but recovery of the Hg²⁺-induced inhibition was not achieved, even when the chelators were present at concentrations above 0.01 M.

Acknowledgements

This study was supported by the Ministry of Science, Technology and Development of the Republic of Serbia, Project No 1991.

Notes

*Hg²⁺, Fe²⁺ and Pb²⁺ don't form complexes with ATP.