Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme

• 1.1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd²⁺ and Zn²⁺ has been studied.
• 2.2. K_s has been determined as 0.14mM. Cd²⁺ and Zn²⁺ were the hyperbolic mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions.
• 3.3. But when the enzyme was inhibited by 0.1 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ reactivated the inhibited form of BChE.

     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from ?130 to ?1,300 kJ/mol), and upon hydration are appreciably lowered.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

Inhibition kinetics of human serum butyrylcholinesterase by Cd2+, Zn2+ and Al3+: comparison of the effects of metal ions on cholinesterases

Butyrylcholinesterase (BChE, EC 3.1.1.8) has been purified about 6600-fold from human serum with a procedure including ammonium sulfate fractionation (55–70%) with acid step at pH 4.5 and procainamide–Sepharose 4B affinity chromatography. The purified enzyme exhibited negative cooperativity with respect to butyrylthiocholine (BTCh) binding at pH 7.5. K_S was found to be 0.128±0.012 mM. Inhibition kinetics of the enzyme by Cd²⁺, Zn²⁺ and Al³⁺ were studied in detail. The 1/v vs 1/[BTCh] plots in the absence (control plot) and in the presence of different concentrations of cations intersected above 1/[BTCh]-axis. The data were analyzed by means of a nonlinear curve fitting program. The results demonstrated that all of the three cations are the linear mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions. But when the enzyme was inhibited by 0.5 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ partially reactivated the inhibited allosteric form of BChE. Results were compared with data obtained from brain BChE purified from sheep.     

Functional characterization of two distinct Mg2+ extrusion mechanisms in cardiac sarcolemmal vesicles

Cardiac ventricular myocytes extrude a sizeable amount of their total Mg²⁺ content upon stimulation by β-adrenergic agonists. This extrusion occurs within a few minutes from the application of the agonist, suggesting the operation of rapid and abundantly represented Mg²⁺ transport mechanisms in the cardiac sarcolemma. The present study was aimed at characterizing the operation of these transport mechanisms under well defined conditions. Male Sprague-Dawley rats were used to purify a biochemical standardized preparation of sealed rat cardiac sarcolemmal vesicles. This experimental model has the advantage that trans-sarcolemmal cation transport can be studied under specific extra- and intra-vesicular ionic conditions, in the absence of intracellular organelles, and buffering or signaling components. Magnesium ion (Mg²⁺) transport was assessed by atomic absorbance spectrophotometry. The results reported here indicate that: (1) sarcolemma vesicles retained trapped intravesicular Mg²⁺ in the absence of extravesicular counter-ions; (2) the addition of Na⁺ or Ca²⁺ induced a rapid and concentration-dependent Mg²⁺ extrusion from the vesicles; (3) co-addition of maximal concentrations of Na⁺ and Ca²⁺ resulted in an additive Mg²⁺ extrusion; (4) Mg²⁺ extrusion was blocked by addition of amiloride or imipramine; (5) pre-treatment of sarcolemma vesicles with alkaline phosphatase at the time of preparation completely abolished Na⁺- but not Ca²⁺-induced Mg²⁺ extrusion; (6) Na⁺-dependent Mg²⁺ transport could be restored by stimulating vesicles loaded with protein kinase A catalytic subunit and ATP with membrane-permeant cyclic-AMP analog; (7) extra-vesicular Mg²⁺ could be accumulated in exchange for intravesicular Na⁺ via a mechanism inhibited by amiloride or alkaline phosphatase treatment; (8) Mg²⁺ accumulation could be restored via cAMP/protein kinase A protocol. Overall, these data provide compelling evidence for the operation of distinct Na⁺- and Ca²⁺-dependent Mg²⁺ extrusion mechanisms in sarcolemma vesicles. The Na⁺-dependent mechanism appears to be specifically activated via protein kinase A/cAMP-dependent phosphorylation process, and can operate in either direction based upon the cation concentration gradient across the sarcolemma. The Ca²⁺-dependent mechanism, instead, only mediates Mg²⁺ extrusion in a cAMP-independent manner.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

Effect of cadmium on ciliary and ATPase activity in the gills of freshwater mussel Anodonta cygnea

1. Cadmium (≤ 50 μM) decreases the heat resistance (39°C) of the activity of frontal cilia in the Anodonta cygnea gills incubated in dechlorinated tap water, while in the presence of added 2 mM Ca²⁺ the minimal acting concentration of cadmium rises up to 100 μM.2. The inhibitory effect of Cd²⁺ (1.5 mM) on the ATPase activity measured in the gill microsomal fraction is temperature dependent and increases as follows: ouabain insensitive Na²⁺- or K⁺-ATPase (no inhibition), Ca²⁺-ATPase (50% inhibition), Mg²⁺-ATPase (100% inhibition).3. Cadmium itself (≤ 50 μM) added to microsomal suspension stimulates the H⁺-sensitive ATP hydrolysis resembling on its pH-dependence the Mg²⁺- but not Ca²⁺-ATPase activity.4. Cd²⁺ can mimic the effect of Mg²⁺ as a cofactor required for activation of the ouabain-insensitive Na⁺- or K⁺-ATPase. Monovalent cations fail to activate the ATPase when Mg²⁺ is substituted by Ca²⁺.5. One of the mechanisms underlying the toxicity of Cd²⁺ to Anodonta gills could be based upon an interaction of Cd²⁺ with Mg²⁺-ATPase followed by suppression of the ciliary activity.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

A new fluorescent chemosensor for Hg2+ in aqueous solution

We prepared an aminothiourea‐derived Schiff base (DA) as a fluorescent chemosensor for Hg²⁺ ions. Addition of 1 equiv of Hg²⁺ ions to the aqueous solution of DA gave rise to an obvious fluorescence enhancement and the subsequent addition of more Hg²⁺ induced gradual fluorescence quenching. Other competing ions, including Pb²⁺, Cd²⁺, Cr³⁺, Zn²⁺, Fe²⁺, Co³⁺, Ni²⁺, Ca²⁺, Mg²⁺, K⁺ and Na⁺, did not induce any distinct fluorescence changes, indicating that DA can selectively detect Hg²⁺ ions in aqueous solution. Copyright © 2012 John Wiley & Sons, Ltd.     

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 (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.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlorotetracycline fluorescence

The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca²⁺ was the major source of fluorescence. In support of this interpretation, incubation in Ca²⁺-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg²⁺ increased it. Raising the Mg²⁺ concentration to 15 mM suppressed the fluorescence. In the presence of Ca²⁺, the substitution of choline ions for Na⁺ increased the fluorescence considerably; in the absence of Ca²⁺, however, Na⁺ deficiency had only little effect. Control experiments showed that Na⁺ or choline ions had no effect on the fluorescence of Ca²⁺-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg²⁺ slightly quenched the fluorescence from 2.5 mM Ca²⁺ and 10 μM chlorotetracycline. It is suggested that Na⁺, and perhaps Mg²⁺, tends to decrease the amount of membrane-bound Ca²⁺ in the pancreatic islets.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Perturbation of Hydrogen Bonds in the Adenine…Thymine Base Pair by Na+, Mg2+, Ca2+ and NH4 + Cations

Abstract

Perturbation of the hydrogen bonds in the adenine…thymine base pair by Na⁺, Mg²⁺, Ca²⁺ and NH₄ ⁺ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine…thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at 06 leads to destabilization of the adenine…thy mine pair; divalent cations (Mg²⁺, Ca²⁺) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.