ATP-dependent 22Na Research Articles

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na+-dependent H+ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na+ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with 22Na+ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na+-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent 22Na+ uptake and abolished the electric potential generated by the Na+-ATPase at the vesicle membrane. A permeant anion NO− 3 accelerated ATP-dependent 22Na+ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO− 3 inhibited the ATP-and Na+-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na+-dependent H+ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na+-ATPase. Hence, the Na+-transporting ATPase of D. maritima carries only one ion species, i.e., Na+. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.

Further evidence for an ATP-driven sodium pump in the marine alga Tetraselmis (Platymonas) viridis

The ATP-dependent 22Na+ accumulation by inside/out plasma membrane vesicles isolated from the marine green microalga Tetraselmis (Platymonas) viridis Rouch. has been studied. The 22Na+ uptake was observed only within a narrow region of weakly alkaline pH with a maxium at pH 7.8–8.0. When permeant anions such as NO3−, were absent from the reaction medium, ATP-dependent 22Na+ uptake was low but could be stimulated by 6 μmol/L ClCCP. In the presence of NO3−, however, the rate of ATP-dependent 22Na+ uptake was much higher than in the absence of nitrate and not affected by the uncoupler ClCCP. The pH optimum of ATP-driven 22Na+ uptake differed from that of ATP-dependent ΔpH formation across the vesicle membranes (optimum pH 6.0–7.0). These data indicate that a ΔμH+-dependent Na+/H+ antiporter does not contribute to the observed uptake under the experimental conditions; 100 μmol/L orthovanadate completely inhibited ATP-dependent 22Na+ uptake, whereas the uptake was not affected by 50 μmol/L amiloride and only slightly reduced by 200 μmol/L of this inhibitor. It is concluded that the ATP-supported 22Na+ uptake by Tetraselmis (Platymonas) viridis plasma membrane vesicles is carried out by a mechanism independent of the proton-motive force. For example it could be catalysed by a primary Na+-pump. It is thought that this pump is an orthovanadate-sensitive electrogenic Na+-ATPase.

Acylphosphatase induced modifications in the functional properties of erythrocyte membrane sodium pump

Human red cell acylphosphatase actively hydrolyzes the Na +/K +-ATPase phosphoenzyme from erthrocyte membrane. This effect occurred with amounts of acylphosphatase (up to 10 units/mg membrane protein) within the physiological range, and the low value of the apparent K m (0.147±0.050 μM) indicates that the enzyme has a high affinity for this substrate. When added at the above concentration to inside out vesicles from human erythrocytes, acylphosphatase significantly enhanced the rate of strophantidine-sensitive ATP hydrolysis. The same amounts of acylphosphatase stimulated, although to a lower extent, the rate of ATP-dependent 22Na + influx (normal efflux). Thus, the calculated stoichiometry for Na +/ATP was 2.68 in the absence of acylphosphatase and 1.06 in the presence of 10 units/mg vesicle protein of the enzyme. Conversely, acylphosphatase addition strongly decreased the rate of ATP-dependent 86Rb + (K +) efflux (normal influx) which, with 10 units/mg vesicle protein, was almost suppressed. As a consequence, the Na +/Rb + ratio, calculated as 1.52 in the absence of acylphosphatase rose to 72.5 in the presence of 10 units/mg vesicle protein of this enzyme. These results suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase ‘uncouples’ erythrocyte membrane Na +, K + pump. Possible mechanisms for this effect are discussed.

Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials. ATP-dependent Nacyt/Kexc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent 22Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Nacyt (2-50 mM) and ATP (1-1000 microM) and pH's (6.5-8.5), the classical 3Nacyt/2Kexc coupling ratio is maintained. However, at low Nacyt concentrations (less than 0.8 mM), the coupling ratio is apparently less than 3Nacyt/2Kexc. ATP-dependent Nacyt/congenerexc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Naexc, act as Kexc surrogates in an electrogenic 3Nacyt/2congenerexc exchange. (ATP + Pi)-dependent Kcyt/Kexc exchange in K-loaded vesicles is electroneutral. ATP-dependent "uncoupled" Na flux into Na- and K-free vesicles is electroneutral at pH 6.5-7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The 22Na flux shows no anion specificity. We propose that "uncoupled" Na flux is an electroneutral 3Nacyt/3Hexc exchange at pH 6.5-7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve Kcyt/Hexc exchange. We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Nacyt in Na/K exchange and Hexc in "uncoupled" Na flux, coupling ratios may change.

Occlusion of cobalt ions within the phosphorylated forms of the Na+-K+ pump isolated from dog kidney.

1. Co2+ ions can replace Mg2+ ions as co-factors for the Na+-K+ pump purified from dog kidney outer medulla. The evidence comes from (a) measurement of ouabain-sensitive Na+,K+-ATPase activity, (b) measurement of ATP-dependent 22Na uptake catalysed by the Na+-K+ pump reconstituted into phospholipid vesicles, (c) measurements of phosphorylation of the Na+-K+ pump either in the presence of ATP and sodium ions or in the presence of inorganic phosphate, and (d) measurement of occlusion of rubidium ions through the route involving phosphorylation and dephosphorylation. 2. Purified Na+,K+-ATPase incubated in the presence of ATP, Na+ ions and [60Co]CoCl2, can carry occluded Co2+ ions through a cation-exchange resin. The enzyme fails to occlude the divalent cation (i) if ADP replaces ATP, (ii) if the enzyme is heat-inactivated, (iii) if the enzyme is inactivated by treatment with fluorescein isothiocyanate, (iv) if K+ replaces Na+ in the incubation medium, (v) if Na+ ions are omitted, and (vi) if Mg2+ ions are added in a sufficient concentration. 3. The amount of occluded Co2+ ions is unaffected by pre-treatment of the Na+,K+-ATPase with oligomycin, which stabilizes the phosphoenzyme in the E1P form. 4. The addition of K+ ions to Na+,K+-ATPase that has been phosphorylated in the presence of ATP, Na+ ions and [60Co]CoCl2 releases the occluded Co2+ ions from the enzyme. Under those conditions, K+ ions accelerate the hydrolysis of the phosphoenzyme, and become occluded in the resulting dephosphoenzyme. 5. The stoichiometry of Co2+ ion occlusion is about one occluded Co2+ ion per phosphorylation site. 6. These results support the hypothesis that, in the normal working of the Na+-K+ pump, Mg2+ ions are trapped in the phosphorylated forms of the enzyme, and are released by a K+-dependent dephosphorylation reaction.

Ammonium ion substitutes for K+ in ATP-dependent Na+ transport by basolateral membrane vesicles.

Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na+-K+-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH+4-containing medium by dilution and centrifugation, and initial rates of 22Na+ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of 22Na+, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of 22Na+ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of 22Na+ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate 22Na+ uptake. Intravesicular K+ could be replaced by NH+4 but not by choline. With NH+4 as counterion, Na+ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K+ and NH+4 on initial rates of 22Na+ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. Our results indicate that ATP-dependent 22Na+ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na+-K+-ATPase, can utilize either K+ or NH+4 as counterion.

Mechanisms of endotoxin-induced impairment in Na+-Ca2+ exchange in canine myocardium.

Effects of endotoxin administration on the Na+-Ca2+ exchange in canine cardiac sarcolemma were characterized. In addition, roles of phospholipase A2 and phosphorylation on the endotoxin-induced impairment in Na+-Ca2+ exchange were investigated. The results show that intravesicular sodium (Nai+)-dependent 45Ca2+ uptake was decreased by 39-66% (P less than 0.05) and extravesicular sodium (Nao+)-induced 45Ca2+ efflux was decreased by 36-52% (P less than 0.05) at 2 h following endotoxin administration. Ca2+o-induced 45Ca2+ efflux in the absence of Ko+ was not affected, but Ca2+o-induced 45Ca2+ efflux in the presence of Ko+ was significantly reduced by endotoxin administration. The stoichiometry of Na+-Ca2+ exchange was altered from 3 Na+ for 1 Ca2+ for the control to 2 Na+ for 1 Ca2+ for the endotoxin-treated dogs. The ATP-dependent 22Na+ uptake and Na+-Ca2+ exchange in inside-out vesicles remained unchanged after endotoxin injection. Digestion of control heart sarcolemmal vesicles with exogenous phospholipase A2 (0.02-0.06 unit/500 micrograms protein) inhibited Na+-Ca2+ exchange activity measured as Nai+-dependent 45Ca2+ uptake, and the inhibition was concentration dependent. Lysophosphatidylcholine addition to the control cardiac sarcolemma had no effect on Na+-Ca2+ exchange. The inhibition of Na+-Ca2+ exchange caused by phospholipase A2 digestion in the control heart sarcolemma was completely reversible by the addition of phosphatidylcholine (0.1 mM). The inhibition of Na+-Ca2+ exchange caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine (0.1-0.15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Uncoupling the red cell sodium pump by proteolysis.

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.

The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump.

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis.

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.

Partial purification and characterization of (Na+ + K+)-ATPase from garfish olfactory nerve axon plasma membrane.

The (Na+ + K+)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na+ + K+)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na+ + K+)-ATPase, such as the Km for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme. The partially purified garfish (Na+ + K+)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent 22Na+ transport. The ratio of 22Na+ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na+ + K+)-ATPase, however, gave a 22Na+ pumped to ATP hydrolyzed ratio of nearly 3.

Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump.

Vesicles isolated from cardiac muscle exhibited Na,Ca exchange activity which can be measured by 45Ca influx or efflux of by 22Na efflux. The stoichiometry of Na,Ca exchange was 3 Na:1 Ca. These vesicles also exhibited ATP-dependent 22Na transport which was inhibited by ouabain indicating that this activity is due to the sodium pump, an activity which is thought to reside only in the sarcolemma. The addition of calcium caused rapid efflux of 22Na from vesicles loaded by ATP-dependent 22Na uptake indicating that the Na,Ca exchange is located in the same vesicles as the sodium pump and is thus also a sarcolemmal activity.

Sodium Transport by Phospholipid Vesicles Containing Purified Sodium and Potassium Ion-activated Adenosine Triphosphatase

Abstract When the purified sodium and potassium ion-activated ATPase (NaK-ATPase) from Squalus acanthias is added to a sonicated mixture of egg lecithin and cholate and dialyzed, vesicles are formed which trap either glucose or inulin. These vesicles are capable of ATP-dependent 22Na+ uptake. Optimum 22Na+ uptake occurs after 2 days dialysis at 4°. 22Na+ uptake was considerably higher at 25° than at 37°, presumably because of a higher lability of the vesicles at the higher temperature. Optimum incubation time for 22Na+ uptake was 1 to 2 hours. The pH profiles for NaK-ATPase activity and 22Na+ uptake were the same. 22Na+ uptake showed a dependence on the presence of Mg2+ but this dependence was not absolute, presumably because of some bound Mg2+ in the preparation. There was a good parallelism in the nucleotide specificity for the NaK-ATPase reaction and 22Na+ uptake, with ATP being the most effective substrate, UTP the poorest substrate, and CTP showing intermediate activity. Internal, but not external, ouabain inhibited 22Na+ uptake, as would be predicted from other observations in the literature which show that ouabain inhibits only on the side of the membrane opposite to the ATP substrate site. The ATP-dependent, ouabain-inhibitable uptake of 22Na+ was shown to be true active transport of Na+, by forming vesicles in the presence of 22Na+ so that the specific activity and concentration of Na+ were the same on both sides of the membrane. In the presence of external ATP there was an actual increase in the concentration of internal Na+ from 20 mm to 38.4 mm. The ratio of Na+ transported to ATP hydrolyzed was 0.3 to 0.4, which is only 10 to 15% of that reported in intact erythrocytes. In the absence of K+, an Na+-Na+ exchange which was dependent on ATP and inhibited by ouabain could be demonstrated; this is similar to Na+-Na+ exchange demonstrated in erythrocytes and muscle. Na+-Na+ exchange was a small percentage of active Na+ transport in the presence of K+. These observations provide strong evidence that vesicles formed from the purified NaK-ATPase of S. acanthias are capable of active sodium transport and that this transport shows the same properties as sodium transport observed in intact cells such as the erythrocyte.