Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits.     

Properties of (Na+ plus K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi.

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124-132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ plus K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ plus K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca(2+), and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ plus K+)-activated ATPase activity averaged 10.07 plus or minus 2.80 mumol Pi/mg protein per h compared to 50.03 plus or minus 11.41 for Mg(2+)-activated ATPase and 58.66 plus or minus 10.07 for 5'-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ plus K+)-activated ATPase without any effect on Mg(2+)-activated ATPase. Both (Na+ plus K+)-activated ATPase and Mg(2+)-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ plus K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ plus K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Rat brain has the alpha 3 form of the (Na+,K+)ATPase

Multiple forms of the catalytic subunit of the (Na+,K+)ATPase have been identified in rat brain. While two of them (alpha 1 and alpha 2) have been well characterized, the third form (alpha 3) of these catalytic subunits only recently has been described by cDNA cloning; the corresponding polypeptide has not been isolated. In this paper it is shown that rat brain contains the alpha 3 chain. The catalytic subunits of the (Na+, K+)ATPase from rat brain axolemma were purified by SDS-PAGE and subjected to formic acid cleavage. Amino acid sequence analysis of the resulting fragments revealed that axolemma has the alpha 3 form of the catalytic subunit. In addition, alpha 3-specific antiserum was raised in rabbits immunized with a synthetic peptide. Immunoblotting with this antiserum revealed that the alpha 3 form of the (Na+,K+)ATPase is present also in whole brain microsomes. In SDS-PAGE, the mobilities of the three catalytic subunits of brain (Na+, K+)ATPase follow the order alpha 1 greater than alpha 2 greater than alpha 3. Determination of the ouabain-inhibitable ATPase activity indicates that if the alpha 3 form of the (Na+,K+)ATPase is able to hydrolyze ATP, it is present in a form of the enzyme with a high affinity for this cardiac glycoside and is similar to the alpha 2 form in this respect.     

Subcellular localization of (Na+ + K+)-activated ATPase in the brush border membrane of the mucosal cell of the rat small intestine.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.     

Detection of a Na+-activated, furosemide-inhibited ATPase activity in rat intestinal mucosa

An ouabain-insensitive, Mg++-dependent, Na+-stimulated ATPase activity which is inhibited by furosemide was found in mucosal homogenate of rat small intestine. The subcellular localization of this ATPase activity was studied by means of isolated purified brush borders and basolateral plasma membranes. The results suggest a nearly identical distribution of Na+-activated and (Na+K+)-activated ATPase within the epithelial cells. Under conditions of alloxan and streptozotocin diabetes an increase of both ATPase activities can be found only in the basolateral plasma membranes. These observations agree well with the convective model of intestinal absorption.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change

The (Na+ + K+)ATPase is inhibited by the bee venom polypeptide, melittin. KCl and NaCl protect the enzyme from melittin inhibition. Analysis of the K+ and Na+ protection against melittin inhibition suggested a kinetic model which was consistent with slowly reversible melittin binding, and mutually exclusive binding of melittin with K+ and Na+. Accordingly, in the absence of salt, the KI for melittin inhibition = 1.2 microM, and the protection by KCl occurs with a KA,KCl = 0.6 mM. The protection by NaCl occurs with a KA,NaCl = 15 mM. Melittin inhibition of enzyme activity is due to direct interactions with the (Na+ + K+)ATPase, as demonstrated by photolabeling with [125I]azidosalicylyl melittin, which labeled the alpha subunit, but not the beta subunit of the (Na+ + K+)ATPase. Melittin and KCl reduced the extent of labeling. In non-covalent binding studies using [125I]azidosalicylyl melittin, the stoichiometry of binding was 1.6 melittin per (Na+ + K+)ATPase. Ligand-induced conformational changes of FITC-labeled (Na+ + K+)ATPase were examined in the presence and absence of melittin. K+ alone or melittin alone caused a fluorescence intensity quenching consistent with formation of an E2 form of the enzyme. The NaCl-induced (E2----E1) fluorescence intensity changes were maximal when the enzyme was treated with K+. NaCl-induced fluorescence changes did not occur when the enzyme was treated with melittin in the absence of K+. However, when K+ was present before the addition of melittin, NaCl-induced fluorescence intensity increases were observed, which were dependent upon the concentration of K+ in the preincubation mixture. The results of the labeling and conformational studies support the kinetic model and suggest a mechanism for inhibition of ion pumps by (poly)peptides.     

Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

Increase of (Na+ + K+)-activated ATPase activity of basolateral plasma membranes from intestinal mucosa of diabetic rats

A significant increase of the (Na+ + K+)-activated ATPase was found in mucosal homogenates of rat small intestine under conditions of alloxan and streptozotocin diabetes. From studies with isolated plasma membranes it has been shown that the activity changes were caused by that part of the (Na+ + K+)-activated ATPase only which is localized in the basolateral plasma membranes, whereas the enzyme activity in the brush border region remains unchanged. In connection with the enhanced capacity of ion, nonelectrolyte and water absorption in experimental diabetes, our findings support a concept of intestinal transport mechanism which suggest that the basolateral part of the (Na+ + K+)-activated ATPase is responsible for metabolic energy supply. The luminal part of the enzyme may be involved in regulation of passive Na+ influx.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport

The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse-chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h).     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Photoaffinity labeling of the ouabain binding site in Na, K-ATPase in developing brine shrimp

Analysis of purified Na,K-ATPase from brine shrimp nauplii by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large (alpha) subunits [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The band with lower mobility in a neutral or alkaline gel is designated alpha 1 and the band with higher mobility alpha 2. Ouabain prevents dephosphorylation of both alpha 1 and alpha 2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of alpha 2. The photoaffinity label, [3H]4'(2-ethyldiazomalonyl) digitoxigenin monodigitoxiside, specifically labels alpha in a ouabain-protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain-protectable labeling of the alpha subunits is associated with alpha 1. The photoaffinity label, [3H]4"' (2-ethyldiazomalonyl) digitoxin, specifically labels alpha 1 and beta in a ouabain-protectable manner without labeling other contaminating proteins. These data show that in the brine shrimp the third digitoxose residue of digitoxin binds in a region in which the alpha 1 and beta chains are in close proximity. Less than 5% of the specific ouabain-protectable labeling of total alpha is associated with alpha 2. These studies indicate that cardioactive steroids have higher affinity for the alpha 1 subunit.     

Modulation of the Na,K-pump function by beta subunit isoforms

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.     

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.     

Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.