Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na⁺ + K⁺)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na⁺ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na⁺ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na⁺ transport sensitively. This technique relies on the presence of Na⁺-Ca²⁺ exchange and ATP-driven Na⁺ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na⁺ uptake is monitored by a subsequent Na_i⁺-dependent Ca²⁺ uptake reaction (Na⁺-Ca²⁺ exchange) using ⁴⁵Ca²⁺. We present evidence that the Na⁺-Ca²⁺ exchange will be linearly related to the prior active Na⁺ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na⁺ isotopes. Applying this method, we measure cardiac ATP-dependent Na⁺ transport and (Na⁺ + K⁺)-ATPase activities in identical ionic media. We find that the (Na⁺ + K⁺)-ATPase and the Na⁺ pump have identical dependencies on both Na⁺ and ATP. The dependence on [Na⁺] is sigmoidal, with a Hill coefficient of 2.8. Na⁺ pumping is half-maximal at [Na⁺] = 9 mM. The K_m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na⁺ pumping. This approach should allow other new investigations on on ATP-dependent Na⁺ transport across cardiac sarcolemma.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

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 K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m 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.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Comparison of kinetic characteristics of Na+ -Ca2+ exchange in sarcolemma vesicles and cultured cells from chick heart

The kinetic characteristics of Na⁺ -Ca²⁺ exchange in isolated sarcolemma vesicles from new-borne chick heart, which contain about 70% of right-side-out vesicles, were compared with those of cultured embryonic chick heart cells. Na⁺ -Ca²⁺ exchange was monitored as Na_i-dependent Ca²⁺ uptake. Increase in the internal concentration of Na⁺ ([Na⁺]_i) in these two preparations caused increase in both the initial rate and the saturation-level of Ca²⁺ uptake. Plots of the rate of Ca²⁺ uptake against [Na⁺]_i showed similar saturation-kinetics in these two preparations. The apparent Michaelis constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) (0.35 mM) for Ca²⁺ uptake by the intact cells was much higher than that (0.031 mM) for Ca²⁺ uptake by the vesicles. The degree of inhibition by Mg²⁺ was also higher in the cells than in the vesicles. Some possible reasons (age of the chicks used, membrane potential, etc.), for these differences were examined and are discussed.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

A microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na⁺ and K⁺ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na⁺ and K⁺. At low Na⁺ concentration (below 5 mM), the apparent K_m decreases with increasing concentrations of K⁺ (1–20 mM). At 5 mM Na⁺, the K⁺ level does not change the apparent K_m, while at Na⁺ levels above 10 mM, the apparent K_m between enzyme and substrate increases with increasing concentration of K⁺. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na⁺ and K⁺. The Na⁺ binding is cooperative with different K_m values and Hill coefficients (n) in the presence of low and high concentration of K⁺. At low Na⁺ level (< 5 mM). a negative cooperativity exists for Na⁺ (n_Na < 1) which is more pronounced in the presence of high [K⁺]. When the concentration of Na⁺ is raised the negative cooperativity disappears and turns into a positive one (n_Na > 1). Only K⁺ binding in the presence of low [Na⁺] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K⁺ (n_K < 1 → n_K > 1). In the presence of [Na⁺] > 10 mM, the changes in n_k are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na⁺, K⁺ and MgATP. 4. In the presence of Na⁺ (< 10 mM), K⁺ is probably bound to two K sites, one of which translocates K⁺ through the membrane by an antiport Na⁺/K⁺ mechanism. This could be connected with an elevated K⁺ uptake in the presence of Na⁺ and could therefore explain some field properties of sugar beets.     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

The influence of cellular amino acids and the Na+: K+ pump on the membrane potential of the Ehrlich ascites tumor cell

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na⁺: K⁺ pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na⁺ but not cellular ATP. Na⁺ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na⁺ and amino acids.Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na⁺ (approx. 30 mM), the Na⁺: K⁺ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na⁺ was raised to 60 mM, the activity of the pump changed the membrane potential from the range ?25 to ?30 mV to ?44 to ?63 mV. This hyperpolarization required external K⁺ and was inhibited by ouabain.