Inhibition of Mg2+ current by single-gene mutation in Paramecium

Eccentric is a newly-isolated mutant of Paramecium tetraurelia that fails to swim backwards in response to Mg²⁺. In the wild type, this backward swimming results from Mg²⁺ influx via a Mg²⁺-specific ion conductance (I _Mg. Voltage-clamp analysis confirmed that, as suspected, step changes in membrane potential over a physiological range fail to elicit I _Mg from eccentric. Further electrophysiological investigation revealed a number of additional ion-current defects in eccentric: (i) The Ca²⁺ current activated upon depolarization inactivates more slowly in eccentric than in the wild type, and it requires longer to recover from this inactivation. (ii) The Ca²⁺-dependent Na⁺ current deactivates significantly faster in the mutant, (iii) The two K⁺ currents observed upon hyperpolarization are reduced by >60% in eccentric. It is difficult to envision how these varied pleiotropic effects could result from loss of a single ion current. Rather, they suggest that the eccentric mutation affects a global regulatory system. Two plausible hypotheses are discussed.We are grateful to Dr. Yoshiro Saimi for his comments and suggestions on this work, and for the support of the Lucille P. Markey Charitable trust and the National Institutes of Health (GM22714 and GM38646).     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

Effect of Cu2+-ascorbic acid on lipid peroxidation,Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu²⁺ and ascorbate in vitro, Mg²⁺ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu²⁺ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg²⁺-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg²⁺-ATPase activity and partially recovers spectrin of RBC membrane.     

Effect of lead on the erythrocyte (Ca2+,Mg2+)-ATPase activity Calmodulin involvement

Summary I have investigated the effect of lead on the erythrocyte ghosts (Ca²⁺,Mg²⁺)-ATPase, with special attention to the role of calmodulin in this phenomena. Under regular incubation conditions, lead inhibits the enzyme with an IC₅₀ of 6.0 µM. The presence of exogenously added calmodulin apparently does not change this inhibitory value. DTT added during the incubation period does not affect the inhibitory action of lead. However, when the membranes are preincubated with DTT, an important IC₅₀ displacement is observed, either with or without added calmodulin. Since [¹²⁵I]calmodulin binding to the membranes is enhanced when lead is used, the possibility of a lead/calmodulin complex that optimally stimulates the enzyme using lead concentrations between 1.0 and 10.0 µM, is suggested. Based on the experimental data, I propose two well defined actions of lead; first, an inhibitory action upon the ATPase above 1.0 µM lead, most probably related to essential sullphydryl groups in the enzyme; and second, a direct action of lead upon calmodulin at lead concentrations below 1.0 µM.A preliminary report has been presented at the 5^th European Bioenergetics Conference. Aberystwyth, Wales. 20–26 July 1988. EBEC Reports. vol 5; p294 (1988).     

Reciprocal effects between spermine and Mg2+ on their movements across the mitochondrial membrane

Mg(2+) competitively inhibits spermine transport in energized rat liver mitochondria (RLM) and exhibits a K(i) of 0.1mM on the initial rate and an I(50) of 0.6mM on total spermine accumulation after 20 min. Addition of 2mM Mg(2+) after spermine accumulation induces release of the polyamine. In view of the fact that spermine cycles across the inner membrane under physiological conditions, these results demonstrate that Mg(2+) inhibits spermine influx but does not affect the efflux pathway of the polyamine; the inhibitory effect occurs via an interaction with the specific site responsible for spermine transport. Instead, spermine inhibits Mg(2+) binding without affecting the rate of Mg(2+) transport, suggesting that both cations bind to the same site, which, however, is not used for Mg(2+) transport. Spermine also inhibits Mg(2+) efflux from RLM induced under conditions of the "low conductance state," a preliminary step preceding permeability transition pore opening.     

Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

We reported previously that a Ca²⁺-ATPase in rat testes and goat spermatozoa could be activated by Ca²⁺ alone without Mg²⁺, though it has a lot of similarities with the well known Ca²⁺, Mg²⁺-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca²⁺ and at low temperature. Increase of the concentration of Ca²⁺ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca²⁺, Mg²⁺-ATPase. The findings strengthened our previous report that Mg²⁺-independent Ca²⁺-ATPase is involved in Ca²⁺ transport and Ca²⁺ uptake like Ca²⁺, Mg²⁺-ATPase.     

Modulation of the activity of the transverse tubule Mg2+ATPase from frog skeletal muscle by a monoclonal antibody in vitro

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109 000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg²⁺-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg²⁺-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.Abbreviations t-tubules transverse-tubules - mAb monoclonal antibody - SR sarcoplasmic reticulum - SL sarcolemma     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Inhibition and stimulation of respiration-linked Mg2+ efflux in rat heart mitochondria

Respiration-driven Mg²⁺ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg²⁺ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg²⁺ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg²⁺ has already been extruded through the energy-linked mechanism. Some inhibition of Mg²⁺ efflux is observed in the presence of high concentrations of La³⁺ (100 µM). A proton/monovalent cation exchanger, nigericin, completely prevents Mg²⁺ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg²⁺ is present in a membrane-bounded compartment, probably in the matrix space. The driving force of the Mg²⁺ efflux appears to be the proton gradient (pH) created by mitochondrial respiration.     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos

Summary During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca²⁺-stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 A/cm² have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca²⁺/Mg²⁺ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca²⁺-stimulated ATPase. The ionic currents and Ca²⁺-stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca²⁺-stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca²⁺-stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase. Offprint requests to: D. Zivkovic     

Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes

Intracellular Mg(2+) buffering and Mg(2+) extrusion were investigated in Xenopus laevis oocytes. Mg(2+) or EDTA were pressure injected and the resulting changes in the intracellular Mg(2+) concentration were measured simultaneously with Mg(2+)-selective microelectrodes. In the presence of extracellular Na(+), injected Mg(2+) was extruded from the oocytes with an estimated v(max) and K(M) of 74 pmol cm(-2)s(-1) and 1.28 mM, respectively. To investigate genuine cytosolic Mg(2+) buffering, measurements were carried out in the nominal absence of extracellular Na(+) to block Mg(2+) extrusion, and during the application of CCCP (inhibiting mitochondrial uptake). Under these conditions, Mg(2+) buffering calculated after both MgCl(2) and EDTA injections could be described by a buffer equivalent with a concentration of 9.8mM and an apparent dissociation constant, K(d-app), of 0.6mM together with an [ATP](i) of 0.9 mM with a K(d-app) 0.12 mM. Xenopus oocytes thus possess highly efficient mechanisms to maintain their intracellular Mg(2+) concentration.     

Light-induced increase in free Mg2+ concentration in spinach chloroplasts: measurement of free Mg2+ by using a fluorescent probe and necessity of stromal alkalinization

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration ([Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast [Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in [Mg(2+)] to 2.0mM in the stroma. The light-induced increase in [Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the [Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the [Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the [Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the [Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg(2+)]. This system for measuring internal chloroplast [Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.     

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg²⁺ concentration ([Mg²⁺]) in chloroplast. Stromal [Mg²⁺] was increased by dark incubation of chloroplasts in the K⁺-gluconate medium (pH 8.0), or by NH₄Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg²⁺] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H⁺ efflux inhibited the alkalinization-induced increase in [Mg²⁺].     

Open-channel block of Na+ channels by intracellular Mg2+

1. Macroscopic and single-channel currents through several types of cloned rat brain Na⁺ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na⁺ channels in chromaffin cells, intracellular Mg²⁺ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na⁺ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg²⁺. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg²⁺ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na] _i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg²⁺ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] _i . This indicates that Mg²⁺ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 10⁸ M^–1 s^–1 and 3.6 · 10⁵ s^–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg²⁺ strongly depends on [Na] _i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg²⁺ block at physiological levels of [Mg] _i .abbreviations [Na]_i intracellular Na⁺ concentration - [K] _i intracellular K⁺ concentration - [Mg] _i intracellular Mg²⁺ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium     

Mg2+ transport in the kidney

Magnesium is abundant in biological systems and an important divalent cation in the human body. Mg²⁺ helps mediate cellular energy metabolism, ribosomal and membrane integrity. Additionally Mg²⁺ modulates the activity of several membrane transport and signal transduction systems. Despite its importance however, little is known about the molecular mechanisms of Mg²⁺ transport and homeostasis in mammals. In mammals the amount of Mg²⁺ absorption is about the same as the amount of Mg²⁺ excretion in urine. Additionally, when total Mg²⁺ intake is deficient, the kidney is capable of reabsorbing all filtered Mg²⁺. This balance between intake and excretion indicates that the kidney plays a principal role in maintenance of total body Mg²⁺ homeostasis. Within the kidney, Mg²⁺ filtered by the glomerulus is handled in different ways along the nephron. About 10–20% of Mg²⁺ is reabsorbed by the proximal tubule. the bulk of Mg²⁺ (about 50–70%) is reabsorbed by the cortical thick ascending limb of the loop of Henle. In this region, Mg²⁺ moves across the epithelium through the paracellular pathway, driven by the positive lumenal transepithelial voltage. A recently cloned human gene, paracellin-1 was shown to encode a protein localized to the tight junctions of the cortical thick ascending limb and is thought to mediate Mg²⁺ transport via the paracellular space of this epithelium. The distal convoluted tubule reabsorbs the remaining 5–10% of filtered Mg²⁺. This segment seems to play an important role in determining final urinary excretion, since there is no evidence for significant Mg²⁺ absorption beyond the distal tubule. Although many renal Mg²⁺ transport activities have been characterized, no Mg²⁺ transporter cDNAs have been cloned from mammalian tissues. Recent research has certainly expanded our knowledge of Mg²⁺ transport in kidney; but details of the transport processes and the mechanisms by which they control Mg²⁺ excretion must await cloning of renal Mg²⁺ transporters and/or channels. Such information would provide new concepts in our understanding of renal Mg²⁺ handling.     

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (Myoglein)

Sarcolemmal Ca²⁺/Mg²⁺ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca²⁺ or Mg²⁺ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca²⁺/Mg²⁺ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca²⁺/Mg²⁺ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca²⁺/Mg²⁺ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.