Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.     

A kinetic analysis of the action of a synaptosomal factor on Na, K-ATPase

A synaptosomal factor stimulated by neurotransmitters activates the Na, K-ATPase system effecting the phosphorylating intermediates moving the Na, K-ATPase system in the mode of simultaneous transport of Na+ and K+. This conclusion has been made during the analysis of kinetics of the effect of MgATP complex, free Mg2+ ions and ATP on Na, K-ATPase activity. Unlike the EGTA, the factor under study does not change the number of essential activators (ions of Na+ and K+) of the Na, K-ATPase system at the equimolar ATP and Mg2+ correlation.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Sodium influence on energy transduction by complexes I from Escherichia coli and Paracoccus denitrificans

The nature of the ions that are translocated by Escherichia coli and Paracoccus denitrificans complexes I was investigated. We observed that E. coli complex I was capable of proton translocation in the same direction to the established deltapsi, showing that in the tested conditions, the coupling ion is the H(+). Furthermore, Na(+) transport to the opposite direction was also observed, and, although Na(+) was not necessary for the catalytic or proton transport activities, its presence increased the latter. We also observed H(+) translocation by P. denitrificans complex I, but in this case, H(+) transport was not influenced by Na(+) and also Na(+) transport was not observed. We concluded that E. coli complex I has two energy coupling sites (one Na(+) independent and the other Na(+) dependent), as previously observed for Rhodothermus marinus complex I, whereas the coupling mechanism of P. denitrificans enzyme is completely Na(+) independent. This work thus shows that complex I energy transduction by proton pumping and Na(+)/H(+) antiporting is not exclusive of the R. marinus enzyme. Nevertheless, the Na(+)/H(+) antiport activity seems not to be a general property of complex I, which may be correlated with the metabolic characteristics of the organisms.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase

Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.     

Functional expression in Escherichia coli of low-affinity and high-affinity Na(+)(Li(+))/H(+) antiporters of Synechocystis

Synechocystis sp. strain PCC 6803 has five genes for putative Na(+)/H(+) antiporters (designated nhaS1, nhaS2, nhaS3, nhaS4, and nhaS5). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na(+)/H(+) antiporter of Pseudomonas aeruginosa, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na(+)/H(+) antiporter of Enterococcus hirae. We successfully induced the expression of nhaS1, nhaS3, and nhaS4 under control of an Na(+)-dependent promoter in Escherichia coli TO114, a strain that is deficient in Na(+)/H(+) antiport activity. Inverted membrane vesicles prepared from TO114 nhaS1 and TO114 nhaS3 cells exhibited Na(+)(Li(+))/H(+) antiport activity. Kinetic analysis of this activity revealed that nhaS1 encodes a low-affinity Na(+)/H(+) antiporter with a K(m) of 7.7 mM for Na(+) ions and a K(m) of 2.5 mM for Li(+) ions, while nhaS3 encodes a high-affinity Na(+)/H(+) antiporter with a K(m) of 0.7 mM for Na(+) ions and a K(m) of 0.01 mM for Li(+) ions. Transformation of E. coli TO114 with the nhaS1 and nhaS3 genes increased cellular tolerance to high concentrations of Na(+) and Li(+) ions, as well as to depletion of K(+) ions during cell growth. To our knowledge, this is the first functional characterization of Na(+)/H(+) antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114 nhaS4 cells did not have Na(+)/H(+) antiport activity, and the cells themselves were as sensitive to Na(+) and Li(+) ions as the original TO114 cells. However, the TO114 nhaS4 cells were tolerant to depletion of K(+) ions. Taking into account these results and the growth characteristics of Synechocystis mutants in which nhaS genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in Synechocystis.     

TNP-8N3-ADP photoaffinity labeling of two Na,K-ATPase sequences under separate Na+ plus K+ control

ATP has high- and low-affinity effects on the sodium pump and other P-type ATPases. We have approached this question by using 2',3'-O-(trinitrophenyl)-8-azidoadenosine 5'-diphosphate (TNP-8N(3)-ADP) to photoinactivate and label Na,K-ATPase, both in its native state and after covalent FITC block of its high-affinity ATP site. With the native enzyme, the photoinactivation rate constant increases hyperbolically with a K(D(TNP-8N)3(-)(ADP)) of 0.11 microM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na(+), but K(+) inhibits with a K(K)' of 12 microM; Na(+) reverses this effect, with a K(Na) of 0.17 mM. This pattern suggests that Na(+) and K(+) are binding at sites in their "intracellular" conformation. It was known that FITC did not abolish the reverse phosphorylation by P(i), or the K(+)-phosphatase activity, and that TNP-8N(3)-ADP could subsequently photoinactivate the latter with >100-fold lower affinity; in that case, the cation sites acted as if facing outward [Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284, 33759-33765]. Native and FITC-modified enzymes have now been photolabeled with TNP-8N(3)-[alpha-(32)P]ADP and alpha-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence alpha-(470)Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys(487), the dropped residue being alphaLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence alpha-(721)Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys(736). These results suggest that Na,K-ATPase also has a low-affinity nucleotide binding region, one that is under distinctive allosteric control by Na(+) and K(+). Moreover, the cation effects seem compatible with a slow, passive Na(+)/K(+) carrier behavior of the FITC-modified sodium pump.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Involvement of Na+/K+pump in fine modulation of bursting activity of the snail Br neuron by 10?mT static magnetic field

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15?min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.     

Electrogenic partial reactions of the gastric H,K-ATPase

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and 相似文献   

Sodium and sulfate ion transport in yeast vacuoles

The intra-luminal acidic pH of endomembrane organelles is established by a proton pump, vacuolar H(+)-ATPase (V-ATPase), in combination with other ion transporter(s). The proton gradient (DeltapH) established in yeast vacuolar vesicles decreased and reached the lower value after the addition of alkaline cations including Na(+). As expected, the uptake of (22)Na(+) was coupled with DeltapH generated by V-ATPase. Disruption of NHX1 or NHA1, encoding known Na(+)/H(+) antiporters, did not result in the loss of (22)Na(+) uptake or the alkaline cation-dependent DeltapH decrease. Upon the addition of sulfate ions, the V-ATPase-dependent DeltapH in the vacuolar vesicles increased, but the membrane potential (DeltaPsi) decreased. Consistent with this observation, radioactive sulfate was transported into the vesicles with a K(m) value of 0.07 mM. The transport activity was unaffected upon disruption of the putative genes coding for homologues of plasma membrane sulfate transporters. These results indicate that the vacuoles exhibit unique Na(+)/H(+) antiport and sulfate transport, which regulate the luminal pH and ion homeostasis in yeast.     

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.     

Effects of Oligomycin on Transient Currents Carried by Na+ Translocation of Bufo Na+/K+-ATPase Expressed in Xenopus Oocytes

Na(+)/K(+)-ATPase (NKA) exports 3Na(+) and imports 2K(+) at the expense of the hydrolysis of 1ATP under physiological conditions. In the absence of K(+), it can mediate electroneutral Na(+)/Na(+) exchange. In the electroneutral Na(+)/Na(+) exchange mode, NKA produces a transient current containing fast, medium and slow components in response to a sudden voltage step. These three components of the transient current demonstrate the sequential release of Na(+) ions from three binding sites. Our data from oocytes provide further experimental support for the existence of these components. Oligomycin is an NKA inhibitor that favors the 2Na(+)-occluded state without affecting the conformational state of the NKA. We studied the effects of oligomycin on both K(+)-activated currents and transient currents in wild-type Bufo NKA and a mutant form of Bufo NKA, NKA: G813A. Oligomycin blocked almost all of the K(+)-activated current, although the three components of the transient current showed different sensitivities to oligomycin. The oligomycin-inhibited charge movement measured using a P/4 protocol had a rate coefficient similar to the medium transient component. The fast component of the transient current elicited by a short voltage step also showed sensitivity to oligomycin. However, the slow component was not totally inhibited by oligomycin. Our results indicate that the second and third sodium ions might be released to the extracellular medium by a mechanism that is not shared by the first sodium ion.     

Ionophore-mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients of methionine by valinomycin in the absence of adenosine triphosphate

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na(+) ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na(+)/l and about 5mequiv. of K(+)/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na(+) and of K(+). Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K(+) progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na(+) was closely coupled to the electrogenic efflux of K(+) through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na(+) and lose K(+). 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na(+) to the amino acid gradient.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport

We have studied the links between the mechanisms of Na(+), K(+) and H(+) movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529-538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na(+) stimulated ATPase activity, K(+) did not affect it, and, secondly, that NN'-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na(+) and H(+) under the experimental conditions applied to the study of the K(+) uptake reported in the preceding paper, and found that when ;Na(+)-loaded cells' previously equilibrated with (22)Na(+) were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of (22)Na(+). This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [(14)C]methylamine distribution indicated that the movement of Na(+) was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na(+)/H(+) antiport. When Na(+)-loaded cells are diluted in an Na(+)-rich medium the Na(+)/H(+) antiport is still active, but cannot decrease the intracellular Na(+) concentration. Under such conditions, net (22)Na(+) extrusion is specifically dependent on the presence of K(+) in the medium. The present results and those derived from the study of K(+) accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na(+) extrusion: an Na(+)/H(+) antiport and an ATP-consuming Na(+)/K(+)-exchange system.     

The conformation of H,K-ATPase determines the nucleoside triphosphate (NTP) selectivity for active proton transport

The gastric H,K-ATPase is related to other cation transport ATPases, for example, Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net ion transport activity. For example, under standard assay conditions, rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP, and UTP. However, we observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs, suggesting that a common phosphoenzyme intermediate is formed. The present study was undertaken to evaluate the specificity of nucleotides to power the H,K-ATPase and several of its partial reactions, including NTP/ADP exchange, K+-catalyzed phosphatase activity, and proton pumping. Results demonstrate that under conditions that promote the conformational change of the K+ bound form of the enzyme, K.E2, to E1, all NTPs tested support K+-stimulated NTPase activity and H+ pumping up to 30-50% of that with ATP. These conditions include (1) the presence of ADP as well as the NTP energy source and (2) reduced K+ concentration on the cytoplasmic side to approximately 0. These data conform to structural models for E1-E2 ATPases whereby adenosine binding promotes the K.E2 to E1 conformational change and K+ deocclusion.     

A novel method to quantify H+-ATPase-dependent Na+ transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na(+) is excluded from the cytosol to the apoplast or the vacuole by Na(+)/H(+) antiporters. The secondary active transport of Na(+) to apoplast against its electrochemical gradient is driven by plasma membrane H(+)-ATPases that hydrolyze ATP and pump H(+) across the plasma membrane. Current methods to determine Na(+) flux rely either on the use of Na-isotopes ((22)Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H(+) gradient due to H(+)-ATPase in the presence or absence of Na(+) by pH-sensitive probes. To date, there are no methods that can directly quantify H(+)-ATPase-dependent Na(+) transport in plasma membrane vesicles. We developed a method to measure bidirectional H(+)-ATPase-dependent Na(+) transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H(+)-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na(+) sealed for the study of H(+)-ATPase-dependent Na(+) transport. Our data implicate that Na(+) movement across vesicle membranes is highly dependent on H(+)-ATPase activity requiring ATP and Mg(2+) and displays optimum rates of 2.50 microM Na(+) mg(-1) membrane protein min(-1) at pH 6.5 and 25 degrees C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H(+)-ATPase-dependent Na(+) transport. The results demonstrate that the Na(+) content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H(+)-ATPase-dependent Na(+) transport with membrane vesicles.