Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Novel streptococcal mutants defective in the regulation of H+-ATPase biosynthesis and in F0 complex

In Streptococcus faecalis (faecium), the cytoplasmic pH is regulated by proton extrusion via a proton translocating F1F0-ATPase; the level of this enzyme increases in response to cytoplasmic acidification (Kobayashi, H., Suzuki, T., and Unemoto, T. (1986) J. Biol. Chem. 261, 627-630). We describe here two novel acid-sensitive mutants, designated AS8 and AS17, that contain ATPase activity but fail to grow on acid media. Our data suggested that in mutant AS17, acidification of the cytoplasm stimulates synthesis of the F0 sector of the ATPase but not the F1 sector. The accumulation in the plasma membrane of F0 sectors devoid of F1 results in enhanced proton permeability, and as a consequence mutant AS17 is unable to regulate the cytoplasmic pH in acid media. The genetic defect may reside in a gene that regulates expression of the F1F0-ATPase. Mutant AS8 does not generate a proton motive force. Our results suggest that the F1F0-ATPase can hydrolyze ATP but fails to translocate protons due to a defect in one of the subunits of the F0 sector.     

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.     

A major Li(+) extrusion system NhaB of Pseudomonas aeruginosa : comparison with the major Na(+) extrusion system NhaP

A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters. We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.     

Effects of pH on the gill ATPase activity and blood composition of whitefish (Coregonus peled) and trout (Salmo trutta fario)

1. The effects of pH on blood composition and gill (Na+ + Mg2+) ATPase activity were studied in 142 whitefish and trout kept for 3 to 17 hr in tanks at pH 3.0-9.5 in Northern Finland. 2. pH clearly influenced all blood values, except plasma alanine and aspartate aminotransferases. Blood haemoglobin and packed cell volume of whitefish and trout were lower at pH 9.5 but higher at pH 3.0 than those of controls at pH 6.5. 3. Both fishes had lowest plasma sodium and chloride values in groups kept for 9 to 17 hr in tanks with water acidified to pH 3.0 to 3.5. Changes in the plasma chloride concentration were associated with plasma sodium (r = 0.92, n = 102, P less than 0.001). 4. The gill (Na+ + K+ + Mg2+) ATPase of trout showed high activity over wide ranges of pH (6.0-7.5) at 13 degrees C having a distinct optimum at pH 7.0. 5. Blood glucose and lactate concentrations of whitefish and trout increased after exposure to acid and base. High values at pH 3.0 to 3.5 suggested hypoxic stress due to acidaemia.     

Bioenergetic properties and viability of alkalophilic Bacillus firmus RAB as a function of pH and Na+ contents of the incubation medium.

The bioenergetic properties and viability of obligately alkalophilic Bacillus firmus RAB have been examined upon incubation in alkaline and neutral buffers in the presence or absence of added Na+. At pH 10.5, cells incubated in the absence of Na+ exhibited an immediate rise in cytoplasmic pH from less than 9.5 to 10.5, and they lost viability very rapidly. Viability experiments in the presence or absence of an energy source further suggested that the Na+-dependent mechanism for pH homeostasis is an energy-requiring function. The Na+/H+ antiporter, which catalyzes the vital proton accumulation at alkaline pH, was only slightly operational at pH 7.0; both whole cells and vesicles exhibited net proton extrusion even in the presence of Na+. Moreover, cells incubated in buffer at pH 7.0 were actually more viable in the presence of Na+ than in its absence. Thus, the inability of B. firmus RAB to grow at neutral pH is not due to excessive acidification of the cytoplasm. Rather, the transmembrane electrical potential, delta psi, generated at pH 7.0 was found to be much lower than at alkaline pH. The very low delta psi compromised several cell functions, e.g., Na+/solute symport and motility, which in this and other alkalophiles specifically depend upon delta psi and Na+.     

Sodium ATPase and sodium/proton antiporter are not obligatory for sodium homeostasis of Enterococcus hirae at acid pH

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na+-ATPase and Na+/H+ antiporter, does not grow in high Na+ medium at pH above 7.5. We found that 7683 grew normally in high Na+ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na+ and K+ were normal in this mutant. The Na+ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na+ elimination of this bacterium at acid pH is achieved by a decrease in Na+ entry and a normal K+ uptake.     

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+-ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore-resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+-ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.0. In the cells grown at pH 7.5, they were moderately observed. The Na+-stimulated ATPase activity of the cells grown at pH 9.5 was about 4-fold higher than that of the cells grown at pH 6.0. Thus, amplification of the Na+-ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.     

Sodium/proton antiporter in Streptococcus faecalis.

Streptococcus faecalis, like other bacteria, accumulates potassium ions and expels sodium ions. This paper is concerned with the pathway of sodium extrusion. Earlier studies (D.L. Heefner and F.M. Harold, Proc. Natl. Acad. Sci. USA 79:2798-2802, 1982) showed that sodium extrusion is effected by a primary, ATP-linked sodium pump. I report here that cells grown under conditions in which sodium ATPase is not induced can still expel sodium ions. This finding suggested the existence of an alternate pathway. Sodium extrusion by the alternate pathway requires the cells to generate a proton motive force. This conclusion rests on the following observations. (i) Sodium extrusion required glucose. (ii) Sodium extrusion was observed at neutral pH, which allows the cells to generate a proton motive force, but not at alkaline pH, which reduces the proton motive force to zero. (iii) Sodium extrusion was inhibited by the addition of dicyclohexylcarbodiimide and of proton-conducting ionophores. (iv) In response to an artificial pH gradient (with the exterior acid), energy-depleted cells exhibited a transient sodium extrusion which was unaffected by treatments that dissipated the membrane potential and which was blocked by proton conductors. I propose that streptococci have two independent systems for sodium extrusion: an inducible sodium ATPase and a constitutive sodium/proton antiporter.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.     

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH.

The Escherichia coli mutant NM81, which is deficient in the nhaA gene for the sodium/proton antiporter, still has a sodium ion extrusion activity because of a second antiporter encoded by nhaB (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). By chance, we have found that E. coli pop6810 already contains a mutation affecting the sodium ion circulation, probably in or near nhaB, and that its delta nhaA mutant, designated RS1, has no sodium ion extrusion activity at alkaline pH. The growth of RS1 was inhibited completely by 0.1 M sodium, whereas growth inhibition of NM81 was observed only at sodium concentrations greater than 0.2 M. RS1 grew at a normal rate in an alkaline medium containing a low sodium concentration. Furthermore, RS1 grew with a negligible proton motive force in the alkaline medium containing carbonyl cyanide m-chlorophenylhydrazone. The transport activities for proline and serine were not impaired in RS1, suggesting that these transport systems could be driven by the proton motive force at alkaline pH. These findings led us to conclude that the operation of the sodium/proton antiporter is not essential for growth at alkaline pH but that the antiporter is required for maintaining a low internal sodium concentration when the growth medium contains a high concentration of these ions.     

Characterization of a Vacuolar Pyrophosphatase in Trypanosoma brucei and Its Localization to Acidocalcisomes

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H(+)-pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 microM) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM), oligomycin (1 microM), N-ethylmaleimide (100 microM), and KNO(3). AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H(+)-ATPase, H(+)-PPase, and (ADP-dependent) H(+)/Na(+) antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H(+)-PPase (both stages) and H(+)/Na(+) exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.     

Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.     

Sodium transport in plant cells

Salinity limits plant growth and impairs agricultural productivity. There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole-plant levels, however, the mechanisms of sodium transport appear to be fundamentally similar. At the cellular level, sodium ions gain entry via several plasma membrane channels. As cytoplasmic sodium is toxic above threshold levels, it is extruded by plasma membrane Na(+)/H(+) antiports that are energized by the proton gradient generated by the plasma membrane ATPase. Cytoplasmic Na(+) may also be compartmentalized by vacuolar Na(+)/H(+) antiports. These transporters are energized by the proton gradient generated by the vacuolar H(+)-ATPase and H(+)-PPiase. Here, the mechanisms of sodium entry, extrusion, and compartmentation are reviewed, with a discussion of recent progress on the cloning and characterization, directly in planta and in yeast, of some of the proteins involved in sodium transport.