Potentiation of 45Ca Uptake and Acute Toxicity Mediated by the N-Methyl-D-Aspartate Receptor: The Effect of Metal Binding Agents and Transition Metal Ions

Abstract: The activities mediated by the N -methyl-D-aspartate (NMDA) receptor were studied in cultured rat cerebellar granule cells. Micromolar concentrations of the metal binding compounds, EDTA, cysteine, and histidine, as well as serum albumin strongly potentiated receptor activity in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺. The findings indicated that these agents remove an endogenous metal, probably Zn²⁺, which attenuates NMDA receptor-mediated ⁴⁵Ca uptake and toxicity. Several added metal ions were therefore tested at low micromolar concentrations. Zn²⁺ was found to be the most potent inhibitor of NMDA-induced ⁴⁵Ca uptake, followed by Cu²⁺ and Fe²⁺. Co²⁺, Cd²⁺, Fe³⁺, and AI³⁺ had no significant effect, whereas Ni²⁺ potentiated the ⁴⁵Ca uptake but inhibited at much higher concentrations. The potentiating agents that remove the endogenous metal had a particularly dramatic effect in the presence of Mg²⁺, the voltage-dependent suppressor of the NMDA receptor. Mg²⁺ also played an important role in the inhibitory effect of added Zn²⁺. Much lower concentrations of Zn²⁺ were needed to achieve inhibition of NMDA-induced ⁴⁵Ca uptake in the presence of Mg²⁺. Under a variety of conditions, a very good correlation was found between NMDA receptor-mediated ⁴⁵Ca uptake and the magnitude of acute neurotoxicity.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

UPTAKE OF MANGANESE BY CHROMAFFIN GRANULES IN VITRO

Abstract– The distribution of metal ions in the chromaffin granule is anomalous. Calcium ions are accumulated but magnesium ions excluded. We have used the manganese ion as a convenient divalent element for following metal ion uptake because it can usefully be employed as a substitute for magnesium.
The kinetics of the manganese incorporation were followed by radio tracer technique and its transport was characterized as being non-energy dependent and having a small thermal activation factor. The presence in the incubation medium of a membrane-bound ATPase inhibitor (NEM) or addition of CN⁻ or NaN₃ did not affect the metal incorporation into chromaffin granules. Little endogenous magnesium exchanges for manganese when the latter is incorporated, but magnesium competitively prevented manganese uptake. The binding of manganese inside the vesicle was strong and it was not exchangeable with magnesium even in the presence of ATP.
Manganese when incorporated binds to ATP and displaces catecholamines resulting in a decrease in the CA/ATP ratio.     

Magnesium Transport by Brain Mitochondria: Energy Requirement and Dependence on Ca2+ Fluxes

Abstract: The association of Mg²⁺ ions with mitochondria isolated from guinea pig cerebral cortex is investigated and resolved into two components, that bound to the surface of both the outer and the inner membranes and that transported into the mitochondrial matrix. When rotenone-treated mitochondria are preincubated in a Mg²⁺ -containing medium, Mg²⁺ binding can be measured and actual Mg²⁺ transport determined after the addition of succinate. Mg²⁺ uptake as well as retention within mitochondria is an energy-dependent process linked to substrate oxidation. EGTA completely prevents Mg²⁺ uptake, while the Ca²⁺ uniporter inhibitor Ruthenium Red, along with prevention of Mg²⁺ uptake, induces a slow efflux of accumulated Mg²⁺ ions. These findings suggest that both inward and outward Mg²⁺ movements follow Ca²⁺ fluxes across the mitochondrial membrane. Modulation of Mg²⁺ movements by mitochondria is therefore suggested to occur within nerve terminals.     

CATION MODULATION OF SYNAPTOSOMAL RESPIRATION

Abstract— Synaptosomes were prepared from the cerebral cortex of the adult rat by a rapid technique, involving the use of centrifugation in a Ficoll-sucrose discontinuous gradient. Adequate respiratory control ratios were obtained with glutamate and succinate plus rotenone. The addition of Na⁺ to the incubation medium stimulated synaptosomal, State-4 respiration, with a half-maximal response at 15 mM Na⁺. The stimulation by Na⁺ was inhibited by atractylate, oligomycin, ouabain or EDTA. A cooperative interaction between Na⁺ and low concentrations of Mg²⁺ was observed. A significant proportion (39 per cent) of the total Na-K ATPase (EC 3.6.1.4) activity in the discontinuous gradient was localized in the synaptosomal fraction. In the absence of exogenous Mg²⁺, Na⁺ induced a 64 per cent stimulation of the synaptosomal ATPase activity which was sensitive to ouabain. Such stimulation of ATP hydrolysis would account for the formation of increased amounts of ADP, with consequent recycling to ATP through adequately controlled oxidative phosphorylation. These observations demonstrate a significant role for transmembrane cationic gradients in the control of synaptosomal respiration and mitochondrial oxidative phosphorylation. The preparation exhibits moderate respiratory control and should prove useful in studies of integrated mitochondrial oxidative metabolism and neuronal membrane function.     

ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg²⁺ or Mn²⁺ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg²⁺ while maximal PI kinase activity required 100 mM Mg²⁺ Similar differences were observed with Mn²⁺ with the exception that maximal ATPase activity occurred with 0.5 mM Mn²⁺ and maximal PI kinase activity occurred with 5 mM Mn²⁺ Mn²⁺ was more effective than Mg²⁺ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg²⁺ was 5-fold greater than the maximal activity obtained with Mn²⁺ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t _l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total ³²P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Calcium-Dependent Binding of Cytosolic Proteins by Chromaffin Granules from Adrenal Medulla

Abstract: Purified chromaffin granules from bovine adrenal medulla bound a small group of medullary cell cytosol proteins at micromolar levels of Ca²⁺ and physiological levels of K⁺, Mg²⁺, and Mg-ATP. The bound proteins had molecular weights of 33,000-37,000 and 70,000-71,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and did not correspond with any previously reported cytosolic components of chromaffin cells. The new proteins were eluted from intact granules or resealed granule membranes at 0.1 μ M Ca²⁺; binding was half-maximal at 2.6 μ M . Adsorption and elution in this manner resulted in a high degree of purification of the new proteins that were minor components of the original cytosol. Partially purified fractions enriched in the 33,000-37,000 and 70,000-71,000 proteins bound ⁴⁵Ca²⁺ at submicromolar levels in the presence of millimolar Mg²⁺. Calmodulin was also bound by the granule membranes and was present in trace amounts in cytosol eluates from granules, but it did not bind to the new proteins in the presence of calcium ions. The possible significance of the new proteins to calcium-mediated secretion from chromaffin cells is discussed.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Pressure-stress effects on the ultrastructure of cells of the dimorphic yeast Candida tropicalis

Abstract Starved cells of cadmium-sensitive Staphylococcus aureus 17810S accumulated ¹⁰⁹Cd via the Mn²⁺ porter energized by the membrane potential (ΔΨ) generated by l-lactate oxidation. However, Cd²⁺ accumulation did not result in inhibition of respiration and consequent generation of electrochemical proton gradient (Δμ_H+) via the respiratory chain. Thus, Δμ_H+-consuming processes, such as ATP synthesis and [¹⁴C]glutamate transport proceeded normally, despite the presence of Cd²⁺ in the cytoplasm. The mechanism of the intrinsic cadmium-insensitivity of the l-lactate oxidizing system is discussed.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Relation of source and sink during grain filling period in wheat and some aspects of its regulation

The effect of Mg²⁺, Na⁺, K⁺, ouabain and pH on ATPase activity of purified membrane fractions enriched in plasmalemma fragments from Hordeum vulgare L. (glycophyte) and Halocnemum strobilaceum L. (halophyte) was studied. Membrane ATPases from both plants were synergistically activated by K⁺ and Na⁺ in the presence of Mg²⁺. The maximum activity of the enzymes were observed at the ratio Na/K = 2–3. Ouabain (10^-4 M) almost completely eliminated the (Na⁺+ K⁺)-stimulated component of the ATPase activity. The Na, K, Mg-ATPase of Hordeum had a single pH optimum (pH 8), but that of the Halocnemum had two optima(pH 6 and 8). It appears that similar enzymes operate in the cells of both plants studied. The higher Na, K, Mg-ATPase activity of the halophyte compared to that of the glycophyte suggests the involvement of the enzyme in the extrusion of Na⁺ from the cytoplasm of cells of both plants.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

Sidedness of (Calcium, Magnesium) Adenosine Triphosphatase of Purified Torpedo Synaptic Vesicles

Abstract: Purified Torpedo synaptic vesicles contain ouabain-insensitive Mg²⁺_τ and Ca²⁺-stimulated ATPase activity. The sidedness of the ATPase on the vesicular membranes was investigated. Addition of ATP and Mg²⁺ or Ca²⁺ to intact vesicles results in activation of the ATPase. Exposure of the vesicles to low concentrations of Lubrol-PX and Triton X-100, which do not solubilize the activity, results in the concurrent release of the vesicular contents and in an increase of the Mg²⁺-ATPase activity, whereas the Ca²⁺-dependent activity is drastically decreased. p -Chloromercuribenzene sulphonate (PCMBS) almost completely inhibits the activity of detergent-treated vesicles whereas that of the native material is only slightly affected. Tryptic digestion of intact vesicles and of vesicular ghosts results in partial reduction of the ATPase activity. These results suggest that the vesicles contain an outward oriented Ca²⁺/Mg²⁺ ATPase activity which can be modulated by detergents.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

Calcium ion uptake by Methanobacterium thermoautotrophicum: Evidence for a carrier-mediated uptake system

Abstract Cell suspensions of Methanobacterium thermoautotrophicum took up ⁴⁵Ca²⁺ in a temperature-dependent, Ca²⁺-saturable and Co²⁺-sensitive process. The accumulation of ⁴⁵Ca²⁺ was lower in the cells energized by CO₂+ H₂ than in those under non-energized conditions. The accumulated Ca²⁺ were, in part, released by the divalent cations ionophore A23187 in the presence of EGTA while the uptake of Ca²⁺ was accelerated by the addition of A23187 to the medium containing Ca²⁺. The results indicate the presence of a carrier-mediated Ca²⁺ uptake in the Methanobacterium thermoautotrophicum membrane which is compensated by an energy-dependent and outward-directed Ca²⁺ transport.