Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.     

Properties of a transplasma membrane electron transport system in HeLa cells

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK _m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.     

Possible Linkage between NADH-Oxidation and Proton Secretion in Zea mays L. Roots

A possible involvement of two different systems in proton translocationand the correlation of this factor to growth rates were measuredsimultaneously by means of a pH stat and an optical system.Ferricyanide, which can accept electrons at the plasmalemma,led to an immediate increase of net H⁺ -efflux but also decreasedroot growth rate. The reduced form, ferrocyanide, inhibitednet H⁺ -effluxwithout changing the growth rate. Thus, corn rootgrowth was not determined by proton secretion exclusively. Vanadatestrongly inhibited net H⁺ -efflux by the roots but did not preventthe stimulating effect of fcrricyanide. Moreover, the extentof enhancement of net H⁺ -effluxby ferricyanide was exactlythe same in vanadate pretreated as in untreated roots. Alcoholswere used to try to increase the intracellular NADH level throughthe action of the cytoplasmic alcohol dehydrogenase presentin the roots and coleoptiles. Alcohols, known to be substratesfor alcohol dehydrogenase such as propan- 1-ol, ethanol andbutan-l -ol increased net H⁺ -effluximmediately but methanoland secondary alcohols which are not substrates had no effecton proton secretion. The K_m values of alcohol dehydrogenasefor the alcohols correspond only partly to their effects onproton secretion. However, the specificlty observed suggeststhat increased H⁺ -efflux arose from reduction of endogenousNAD by ADH and consequent increased membrane NADH-oxidasc activitytrans locating protons and electrons out of the cells. Decreased oxygen concentrations slowed proton secretion at valuesfar higher than are necessary to saturate cytochrome c oxidase.The results of these experiments suggest two distinct systemscontributing to proton efflux. Key words: ADH, proton transport, redox chain     

Serine metabolism in legume nodules: Purification and properties of phosphoserine aminotransferase

Plant cells excrete protons via an electrogenic proton pump, the K⁺-stimulated, Mg²⁺-dependent H⁺-ATPasc, located on the cytoplasmic side of the plasma membrane. Plasma membrane redox reactions are also coupled to proton excretion. Various inhibitors were used on carrot ( Daucus carola L.) cells in an attempt to distinguish between the two processes. Inhibitors of electron transport reactions in the plasma membrane (chloroquine, 8-hydroxyquinolinc, 4,7-dichloroquinolinc and retinoic acid) inhibited ferricyanide-induced proton excretion by 37–100%, while they inhibited potassium ferricyanide reduction, a measure of plasma membrane redox activity, by 42–100%. The above-mentioned quinolines and retinoic acid inhibited cell growth by 49–98%, with the exception of chloroquine, which stimulated carrot cell growth by 36%.     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca²⁺ and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP⁺, SDS^–) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.Abbreviations CP⁺ cetylpyridinium - EGTA ethylene glycol bis (-aminoethyl)-N,N-tetraacetic acid - FeCN potassium ferricyanide - SDS^– sodium dodecyl sulfate - SHAM salicylhydroxamic acid     

Mammalian Complex I Pumps 4 Protons per 2 Electrons at High and Physiological Proton Motive Force in Living Cells

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H⁺/2e⁻) but it has been suggested that stoichiometry may be 3H⁺/2e⁻ based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD⁺ and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc₁ complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H⁺/2e⁻ in mouse and human cells at high and physiological proton motive force.     

Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H⁺-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H⁺-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H⁺-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H⁺-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H⁺-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.     

Changes in lipid peroxidation, the redox system and ATPase activities in plasma membranes of rice seedling roots caused by lanthanum chloride

Highly purified plasma membranes were isolated by aqueous two-phase partitioning from rice (Oryza sativa) seedling roots. The effects of lanthanum chloride (LaCl₃) on the activities of lipid peroxidation, the redox system and H⁺-ATPase, Ca²⁺-ATPase of plasma membranes were studied. The lipid peroxidation of plasma membranes could be depressed by certain low concentrations of LaCl₃ and enhanced by high concentrations of LaCl₃, while the lipid peroxidation was also dependent on the plasma membrane protein and incubation time. The relative activity of O₂ uptake of plasma membranes was inhibited by all tested LaCl₃ concentrations. In contrast, the reduction rate of Fe(CN)₆ ^3– by plasma membranes was stimulated below 40 M of LaCl₃, but was reduced above 60 M of LaCl₃. The relative activities of both H⁺-ATPase and Ca²⁺-ATPase increased constantly from control to LaCl₃ of concentration 60 M where the activities of both enzymes were the maximum, but decreased remarkably at 80 M LaCl₃ concentrations various LaCl₃ were added to culture solutions. In the other measurement case in which various LaCl₃ concentrations were added directly to reaction medium and the plasma membrane vesicles only came from the control cultured rice seedling roots, the response of H⁺-ATPase activity to La³⁺ was similar to the response in culture solution. However, the La³⁺ concentration was only 20 M when the activity of H⁺-ATPase was the maximum. In contrast to the case of LaCl₃ addition to culture solution, Ca²⁺-ATPase activity was inhibited by all concentrations of La³⁺ which were added directly to the reaction medium. The above results revealed that REEs inhibited electron transfer from NADH to oxygen in plant plasma membranes, depressed the production of active oxygen radicals, and reduced the formation of lipid peroxides through plasma membrane lipid peroxidation. REEs ions also enhanced the H⁺ extrusion by both standard redox system and H⁺-ATPase in plasma membranes at certain concentrations. A possible role for the plant cell wall in REEs effects on plasma membranes was also suggested.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

On the mechanism of H+ translocation by mitochondrial H+-ATPase. Studies with chemical modifier of tyrosine residues

In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H⁺ conduction and other reactions catalyzed by the H⁺-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-³³P_i exchange, and proton conduction by the H⁺-ATPase complex. These effects are caused by nitration of tyrosine residues of H⁺-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-³³P_i exchange in the complex purified from tetranitromethane-treated particles. H⁺ conduction in phospholipid vesicles inlaid with F₀ is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F₀ are critically involved in energy-linked proton translocation in the ATP-ase complex.     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.