The effects of Ni2+, Co2+, and Mn2+ on human serum butyrylcholinesterase

The effects of Ni2+, Co2+, and Mn2+ on human serum butyrylcholinesterase (BChE, acylcholine acylhydrolase E.C. 3.1.1.8) were investigated in this study. Inhibition kinetics of BChE were studied using butyrylthiocholine (BTCh) as substrate. The "1/v" versus "1/[BTCh]" plots in the absence (control plot) and in the presence of the metal ions intersected above 1/[BTCh]-axis for all trace elements. In addition, when the concentrations of the cations were increased at 4 mM BTCh, velocities decreased and drove to zero at high concentrations of the trace elements. These results demonstrate that Ni2+, Co2+, and Mn2+ are linear mixed-type inhibitors of BChE. alphaK(i) values have been determined as 53.20 mM,152.25 mM, and 190.24 mM for Ni2+, Mn2+, and Co2+, respectively, by using nonlinear regression analysis. From the comparison of alphaK(i) values of the trace elements, it can be said that BChE has more affinty to binding Ni2+ than Co2+ and Mn2+.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

Effect of bivalent metal ions on the fluctuations of ion currents in rat liver mitochondria

Ions of bivalent metals are shown to arrange in the Sr2+ greater than Ca2+ greater than Ba2+ greater than Mn2+ series as to their ability to induce ion flow vibration in the rat liver mitochondria. Application of Sr2+ results in the most stable prolonged vibrations of ion flows in mitochondria. Ca2+, Ba2+ and Mn2+ induce slightly pronounced and intensively damped vibrations. The studied Mg2+, Co2+, Ni2+, Pb2+ Fe2+ cations have effect on valinomycin-induced K+ transport in mitochondria and do not induce vibrations. It is established that the ability of bivalent cations to induce vibrations is associated with the possibility of their transfer through the mitochondrion membrane and accumulation in the matrix. Inhibitors of the electrogenic Ca2+ transport in mitochondria produce the similar effect on vibrations induced by Sr2+, Ca2+, Ba2+ and Mn2+.     

Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions.

The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.     

Effects of bivalent cations on post-rest adaptation in guinea-pig heart muscle

In isolated papillary muscles of guinea-pig hearts, the inotropic effects of bivalent cations, Ca2+, Ba2+, Sr2+, and Ni2+, were investigated during post-rest adaptation in order to study their individual action on excitation-contraction coupling. Upon exposure to each cation studied, the force of contraction was transiently enhanced, whereas the steady state force was influenced differently: it increased with Ca2+, Ba2+ and Sr2+ and was depressed by Ni2+. The transmembrane action potentials (measured at 90% repolarization) were slightly prolonged by Sr2+ and even more by Ba2+, and were shortened by Ca2+ and Ni2+. After 10 min rest, the post-rest contractions consisted of a late peak (PII) that was enhanced in high Ca2+-solution an by Sr2+. Ni2+ and Ba2+ depressed PII and during adaptation to pre-rest controls an early peak of contraction (PI) prevailed. There was no simple relation between post-rest adaptation of force and the duration of action potential in the presence of the bivalent cations tested. During post-rest adaptation the two components of contraction can be separated. The results are interpreted in terms of a model of excitation-contraction coupling which derives Ca ions for contractile activation from two sources: transmembrane calcium influx and calcium release from cellular stores. From the different effects on post-rest adaptation it is concluded that the individual cations influence excitation-contraction coupling more specifically and not merely by "screening-off" the negative surface charges.     

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.     

Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris)

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709- 728). This influx was blocked by multivalent cations with half- inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.     

Interaction of EGTA with a hydrophobic region inhibits particulate adenylate cyclase from rat cerebral cortex: a study of an EGTA-inhibitable enzyme by using alamethicin

Washed membranes isolated from rat cerebral cortex (gray matter) showed the presence of EGTA-inhibitable and EGTA-insensitive forms of adenylate cyclase activity. The former activity was stimulated by low concentrations (microM) of various divalent cations (Mn2+, Ca2+, Co2+ and Sr2+) assayed with MgATP2- and MgCl2. At higher concentrations (mM), only Mn2+ stimulated this enzyme whereas Ca2+, Co2+ and Sr2+ were inhibitory. Alamethicin markedly (up to 30-fold) increased the activity of EGTA-inhibitable form and only moderately of EGTA-insensitive form of the enzyme. The increased activity due to alamethicin does not result from solubilization of the enzyme from membranes. Our results suggest the presence of two distinct metal binding sites--one of high (Site I) and other of low (Site II) affinity. Divalent metals via interacting with these produce divergent effects on the enzyme. Site I appears to be located in the hydrophobic region of catalytic unit of the enzyme or of membrane-associated calmodulin. The likely significance of these results is briefly presented.     

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.     

Role of cations in the flocculation of Saccharomyces cerevisiae and discrimination of the corresponding proteins

Asexual yeast flocculation was studied using strong flocculents of Saccharomyces cerevisiae. The inhibitory effect of cations on flocculation is considered to be caused by competition between those cations and Ca2+ at the binding site of the Ca(2+)-requiring protein that is involved in flocculation. Inhibition of flocculation by various cations occurred in the following order: La3+, Sr2+, Ba2+, Mn2+, Al3+, and Na+. Cations such as Mg2+, Co2+, and K+ promoted flocculation. This promoting effect may be based on the reduction of electrostatic repulsive force between cells caused by binding of these cations anionic groups present on the cell surface. In flocculation induced by these cations, trace amounts of Ca2+ excreted on the cell surface may activate the corresponding protein. The ratio of Sr2+/Ca2+ below which cells flocculated varied among strains: for strains having the FLO5 gene, it was 400 to 500; for strains having the FLO1 gene, about 150; and for two alcohol yeast strains, 40 to 50. This suggests that there are several different types of cell surface proteins involved in flocculation in different yeast strains.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.