Discrimination between Alkali Metal Cations by Yeast : I. Effect of pH on uptake

Extracellular pH markedly influences the ability of yeast cells to discriminate between K⁺ and Na⁺, with K⁺ favored to a greater degree at low pH. Studies of the kinetics of uptake of individual alkali metal cations by fermenting yeast indicate three zones relative to pH. Between pH 6 and 8, H⁺ has no effect. Below pH 4, H⁺ competitively inhibits the transport of each cation. Between pH 4 and 6, H⁺ acts kinetically as a predominantly non-competitive inhibitor. Both effects can be reversed by increasing the concentrations of cations. However, the concentrations required to reverse the competitive effect are considerably lower than those required to reverse the apparently non-competitive effect. It is suggested that H⁺ and the alkali metal cations can combine with two sites, a transport or carrier site, and a second, non-transporting site that influences the maximal rate of transport. Because the non-competitive inhibitory effect of H⁺ is considerably greater on the other cations than on K⁺, the discrimination in favor of K⁺ is increased severalfold at low pH, beyond that predicted on the basis of the relative affinities for the transport site.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).     

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.     

Interrelationships between Potassium, Calcium, and Magnesium Nutrition of Zea mays L.

The uptake of potassium, calcium, and magnesium ions by maizeand the interrelationships among the cations have been investigatedat 48 K: Ca: Mg ratios in culture solutions. Calcium was foundto stimulate K⁺ and Mg⁺⁺ uptake at certain cation ratios butinhibit it at others. Potassium did the same for Ca⁺⁺ uptake,and Mg⁺⁺ for Ca⁺⁺ and K⁺. The uptake of Mg⁺⁺ was generally enhancedby K⁺. The sum of the cations in the plants expressed in meqwas fairly constant for treatments of the same K⁺ concentrationat the low to moderate levels of K⁺, but at considerably higher(> 24 meq l^–1) K⁺ levels the constancy was not dependenton K⁺ concentration. Potassium depressed, but Mg⁺⁺ stimulatedphosphorus accumulation. Calcium stimulated phosphate absorptionat certain cation ratios but had no effect at others. The plantyield increased with increasing K⁺ up to 24 meq l^–1 ofK⁺ after which the yield tended to fall with further increasein K⁺. The yield was also increased by Ca⁺⁺. Magnesium increasedthe yield at certain cation ratios and either depressed it orwas without effect at others.     

Thallium interaction with the gastric (K,H)-ATPase

Summary The gastric (K,H)-ATPase has been shown to catalyze an electroneutral H⁺ for K⁺ exchange. Tl⁺ is able to substitute for K⁺ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 m as compared to about 870 m for K⁺. The ability of Tl⁺ to participate in transport is shown by the development of pH gradients in the presence of Tl⁺ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl⁺ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl⁺ concentrations above 3.0mm. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl⁺ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl⁺ concentrations, following the initial Tl⁺ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl⁺ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t _1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl⁺ concentrations in excess of 1 to 4mm are able to dissipate intravesicular proton gradients by an electrically coupled H⁺ for Tl⁺ exchange. Thus, although Tl⁺ is able to activate the gastric ATPase by mimicking K⁺, the permeability of this cation in lipid bilayers tends to uncouple H⁺ transport at concentrations high enough to generate detectable proton gradients.     

Interaction of ethidium bromide with the transport system for monovalent cations in yeast

Summary Ethidium was found to be taken up by yeast cells in a process that, at certain concentrations has the main following characteristics: a) a substrate is required; b) it presents cooperative kinetics, withn, according to the Hill equation 3; c) ethidium can be concentrated more than 100-fold; d) the uptake is inhibited by Ca²⁺; e) the uptake of the dye is inhibited by monovalent cations with a selectivity pattern similar to that observed in their transport by yeast; f) ethidium inhibits the uptake of K⁺, and, at concentrations up to about 250 m produces a competitive inhibition on the uptake of Rb⁺; and g) ethidium produces the same effects as K⁺ on respiration and the extrusion of H⁺. It is concluded that ethidium is taken up by yeast cells in a selective way by the same transport system normally employed for monovalent cation uptake.     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.     

Activation of wheat germ acetyl CoA carboxylase by potassium and rubidium.

Wheat germ acetyl CoA carboxylase requires certain alkali cations to exhibit maximal activity. Maximal activation results when 60 mM K⁺ or Rb⁺ are included in the assay mixture, whereas only marginal activation occurs in the presence of similar concentrations of Li⁺⁺ and Na⁺⁺. Cs⁺⁺ activates, but less effectively than K⁺ or Rb⁺. Since it is also possible to activate the enzyme maximally using 20 mM potassium isocitrate, but not 20 mM sodium isocitrate, activation of the wheat germ enzyme is due to a cation effect and not to citrate anion.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Proton permeability and the regulation of potassium permeability in mitochondria by uncoupling agents

Summary The addition of agents that uncouple electron transfer from energy conservation (uncouplers) to state 4 mitochondria causes the following ion movements: K⁺ is extruded from the mitochondria in association with phosphate and possibly other anions, but not H⁺. Endogenous Ca⁺⁺ is extruded from the mitochondria, and H⁺ moves in to counter-balance the Ca⁺⁺ movement; some phosphate movement may be associated with Ca⁺⁺ extrusion. The rate and extent of K⁺ extrusion induced by uncoupler is dependent on the concentrations of external phosphate and divalent ions. Phosphate induces K⁺ extrusion, while Mg⁺⁺ and Mn⁺⁺ inhibit it. TheV _max of K⁺ transport is 300 moles K⁺/g protein per min. The K _m for FCCP-induced potassium extrusion is 0.25 M at pH 7.4. The inhibitory effect of Mg⁺⁺ is noncompetitive with respect to uncoupler concentration but competitive with respect to phosphate concentration. The experimental evidence does not support the existence of high H⁺ permeability in the presence of uncoupler. A correlation is observed between the rate of K⁺ extrusion and the energy reserves supplied from the high energy intermediate. The action of uncoupler in inducing K⁺ permeability is considered to arise through its action in depleting the energy reserves of mitochondria rather than through a specific activating effect of permeability by the uncoupler itself. The relationship of membrane potential to regulation of K⁺ permeability is discussed.     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

Studies on the mechanism of K+ transport in yeast.

The effect of uncouplers and diffusible acids on K⁺ transport was studied in yeast.Although the K⁺ transport system seems to depend on ATP to function, the effects of uncouplers are not due primarily to its action on the energy conserving systems of the cell.Other uncouplers with different structures to that of DNP showed also an inhibitory effect on K⁺ transport, which agrees with their reported ability to conduct protons through membranes.Uncouplers, besides inhibiting K⁺ uptake, produce an efflux of this cation; however, the rate of efflux produced is quantitatively important only when the cells have previously taken up the cation; there seems to exist a mechanism which prevents the loss of cations by yeast.In the absence of substrate, at pH 8.5, with 0.5 m KCl, TCS produces the efflux of H⁺, and when ⁸⁶Rb⁺ was used as a substitute for K⁺, an increase of the entrance of the cation could be detected in the presence of the uncoupler. It seems that the effect of the uncoupler depends on the direction of the combined H⁺ and K⁺ gradients, or the electrochemical potential of the cell.As reported by other authors, weak diffusible acids increase the uptake of K⁺ by yeast, and this effect is not due to changes in the metabolism, but to the magnitude of the entrance of the molecules to the yeast cell.It was found that the efflux of the acids (H₂CO₃), on the other hand, can produce an efflux of K⁺, which means that anions are important not only for the entrance of the cations, but for its permanence within the cell as well.The data seem to be in agreement with the hypothesis of the existence of a proton pump, responsible for the creation of an electrochemical potential, involved in K⁺ transport. At low pH, this pump seems to be activated by the transport of K⁺ into the cell.     

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts

Summary The exposure of red cell ghosts to external Ca⁺⁺ and K⁺ leads to a rapid net K⁺ efflux. Preincubation of the ghosts for various lengths of time in the absence of K⁺ in the external medium prior to a challenge with maximally effective concentrations of Ca⁺⁺ and K⁺ renders the ghosts unresponsive to that challenge with a half-time of about 7–10 min. Preincubation at a range of K⁺ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K⁺ concentrations of about 500 m or more suffice to maintain the K⁺ channel in a maximally responsive state for at least 60 min. These K⁺ concentrations are considerably lower than the K⁺ concentrations required to make the responsive channel respond with a maximal rate of K⁺ efflux. Thus, external K⁺ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state.The presence of Mg⁺⁺ or ethylenediamine-tetraacetic acid (EDTA) in the K⁺-free preincubation media preserves the responsiveness to a challenge with Ca⁺⁺ plus K⁺. In contrast to external K⁺, the presence of external Ca⁺⁺ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca⁺⁺ while washes with EDTA after exposure to Ca⁺⁺ do not reverse them.In red cell ghosts that contain Ca⁺⁺ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K⁺ is enhanced. The effects of incubation in the presence of Ca⁺⁺ in K⁺-free media are reversed; external Ca⁺⁺ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K⁺-free media prior to a challenge with external K⁺ and internal Ca⁺⁺ does also take place when K⁺-efflux from red cell ghosts is measured by means of⁴²K⁺ into media that have the same K⁺ concentrations as the ghost interior. This confirms that the effects of K⁺-free incubation are due to the modification of the K⁺-selective channel rather than to an inhibition of diffusive Cl^–-efflux.Abbreviation used in text TRIS Tris (hydroxymethyl) aminomethan This paper is dedicated to the memory of Walther Wilbrandt.     

Facilitated transport of di- and trinitrophenolate ions across lipid membranes by valinomycin and nonactin

The conductance of black lipid membranes in the presence of 2,4,6-trinitrophenol (or 2,4-dinitrophenol) is considerably enhanced, if the cation carriers valinomycin, enniatin B or nonactin are added. The effect is, however, largely independent of the cation concentration and is identical for the cations Li⁺, Na⁺ and Ba²⁺. This finding, as well as the sign and magnitude of the diffusion potential in the presence of a gradient of picrate are consistent with the assumption that the transport of picrate anions is facilitated by the above-mentioned macrocyclic compounds, but that cations are not directly involved. A model is suggested which, based on the generation of mobile defect structures by the incorporation of large molecules, allows one to explain facilitated transport without the assumption of stable chemical bonds between a carrier and its transported substrate.If K⁺ is present in the aqueous phase, the conductance is largely determined by the permeation of the cation complexes of valinomycin and nonactin. The conductance is, however, increased by adsorption of picrate anions to the membrane surface. The negative surface potential generated by the adsorption layer seems to be responsible for the saturation of the conductance at high picrate concentrations in the absence of valinomycin and nonactin.     

Uptake and effects of several cationic dyes on yeast

Summary Several cationic dyes were found to behave as inhibitors of K⁺ uptake in yeast. When added at high concentrations or in a K⁺-free medium, dyes can also produce and efflux of K⁺. The dyes are taken up by the cells in a process that, in different degrees, for several cations requires glucose and is inhibited to a higher degree by K⁺ than by Na⁺.The inhibition of cation uptake is of the competitive type with EB and close to this type with other dyes. Ca²⁺ inhibits the uptake and effects of dyes and in some cases also seems to change the inhibition kinetics on Rb⁺ uptake closer to a pure competitive type.According to preliminary experiments, the efflux of K⁺ seems to be of the electrogenic type, and not due to the disruption of the cells. The data indicate that, independently of the existence of other types of interaction (which do exist), dyes seem to interact with the system for monovalent cation uptake of yeast in different degrees of specificity and energy requirement. This interaction can be followed by fluorescence or metachromatic changes or reduction of the dyes as observed in the dual wavelength spectrophotometer and can be inhibited specifically by K⁺, but not by Na⁺.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.