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.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G _c ^min ) of the transcellular conductance, estimated from Ba⁺⁺ blockade of apical membrane K⁺ channels, indicated thatG _c ^min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K⁺ was the major conductive species; and ADH increased the magnitude of a Ba⁺⁺-sensitive K⁺ conductance under conditions where net Cl^– absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl^– conductance; this ADH-dependent increase in basal Cl^– conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl^– absorption. Cl^– removal from luminal solutions had no detectable effect onG _e , and net Cl^– absorption was reduced at luminal K⁺ concentrations less than 5mm; thus apical Cl^– entry may have been a Na⁺,K⁺,2Cl^– cotransport process having a negligible conductance. The net rate of K⁺ secretion was approximately 10% of the net rate of Cl^– absorption, while the chemical rate of net Cl^– absorption was virtually equal to the equivalent short-circuit current. Thus net Cl^– absorption was rheogenic; and approximately half of net Na⁺ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K⁺ was the principal conductive species in apical plasma membranes, support the view that the majority of K⁺ efflux from cell to lumen through the Ba⁺⁺-sensitive apical K⁺ conductance pathway was recycled into cells by Na⁺,K⁺,2Cl^– cotransport.     

[14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open

Following small hypo-osmotic shocks, ion concentrations (Na⁺, K⁺, Cl^-) in Platymonas subcordiformis decreased; this was due mainly to an increase of cell volume. With larger hypo-osmotic stresses, the decrease of ion concentration continued and, additionally, extrusion of mannitol was observed. The ion and mannitol concentrations were not regained after 240 min. In contrast, following hyperosmotic shocks, the ion concentrations increased transitorily during the first 20–40 min. The same was true for K⁺ following small hyperosmotic stresses and for Na⁺ and — partially — Cl^- with larger shocks. Large hyperosmotic stresses caused permanent accumulation of mannitol, which levelled off after 60–80 min. Thus the transient increase of ions bridged the concentration gap until mannitol was accumulated to a high enough concentration to account for the osmotic adaptation of Platymonas, together with a basal level of the ions K⁺, Na⁺, Cl^-.Abbreviations PS photosynthesis - Resp respiration     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

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⁺⁺.     

A study of sodium release in the course of ATP hydrolysis by membrane ATPase

Summary With the aid of sodium-sensitive glass electrodes, changes in sodium ion activity were studied in the course of subsequent additions of components required for ATP hydrolysis provided by Na⁺–K⁺-dependent membrane ATPase. Membrane ATPase was obtained from guinea pig kidney cortex. In the presence of ATP, Mg⁺⁺ and Na⁺ in media, the addition of K⁺ caused an increase in Na⁺ activity. The omission of ATP or its substitution by ADP as well as the addition of Ca⁺⁺ to the media eliminated the above-mentioned increase of Na⁺ activity. Quabain did not affect Na⁺ release caused by the addition of K⁺, although it significantly inhibited ATPase activity of the preparation. The data obtained were considered to be a direct indication of ion exchange during the course of membrane ATPase reaction. This ion-exchange stage of the reaction is not inhibited by ouabain. The ratio of sodium ions released per one inorganic phosphate formed in the course of the reaction was found to be much higher than that established for transporting membranes of intact cells. A possible cause of this difference is discussed.     

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

Cation co-tolerance phenomenon in cell cultures of Oryza sativa adapted to LiCl and NaCl

Cell lines of Oryza sativa L. (cv. Taipei-309) were adapted to 30 mM LiCl and 150 mM NaCl. Both adapted lines were considerably more tolerant than non adapted line when grown on 200, 250 and 300 mM NaCl and 30 mM LiCl stresses. The tolerance of LiCl-adapted line to NaCl (150 to 300 mM) and the tolerance of NaCl-adapted cells line to LiCl (30 mM) indicated that there was a cross-adaptation towards alkali metals (Na⁺ and Li⁺) not the Cl^–. Na⁺ and K⁺ contents of all lines which increased with increasing medium salinity but to a different degree. The increase in Na⁺ and K⁺ content in NaCl-adapted and non-adapted lines were comparable, while LiCl-adapted line accumulated significantly lower Na⁺and higher K⁺ content. Proline content of all lines increased with the increase in NaCl-stress but the magnitude of increase was much higher in the LiCl-adapted than other lines. The differential response of adapted lines to NaCl stress in accumulating proline and maintaining the ionic contents reveals that adapted lines have evolved different features of adaptation to cope with NaCl stress.     

Ionic mechanisms of nonlinearity of the retinal horizontal cell membrane

Changes in ionic conductance lying at the basis of nonlinearity of the current-voltage characteristic curve of the cell (nonsynaptic) membrane of horizontal cells were studied in experiments on the goldfish and turtle retina. All measurements were made during blocking of synaptic transmission by bright light or Co⁺⁺. An increase in the K⁺ concentration led to depolarization and to a reduction of the steepness of the hyperpolarization branch of the current-voltage curve, whereas a decrease in K⁺ had the opposite effect. Changes in the Cl^– or Na⁺ concentrations had no significant effect on membrane potential or on the shape of the current-voltage curve. The principal potential-forming ion in the horizontal cells is thus K⁺; conductance for Cl^– is absent or very low, and conductance for Na⁺ also is evidently small. In the presence of Ba⁺⁺ (2–5 mM) the steepness of the hyperpolarization branch of the current-voltage curve was increased and the whole curve became more linear. It is concluded that nonlinearity of the current-voltage curve of the horizontal cell membrane is due mainly to potential-dependent potassium channels, whose conductance increases during hyperpolarization; this increase in conductance is blocked by Ba⁺⁺. An increase in the Ca⁺⁺ concentration to 20 mM led to an increase in steepness of the depolarization branch of the current-voltage curve, suggesting that depolarization increases membrane conductance for Ca⁺⁺.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 531–539, September–October, 1981.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Ion metabolism in a halophilic blue-green alga,Aphanothece halophytica

The intracellular ion content of the halophilic blue-green alga, Aphanothece halophytica was studied as a function of age, external sodium and external potassium concentration. Intracellular Na⁺ was found to be about 0.38 millimoles/g dry mass. Intracellular K⁺ concentrations were as high as 1 M and varied directly with external salinity. Intracellular Ca⁺⁺ and Mg⁺⁺ were in the range previously reported for fresh water blue-green algae despite their extremely high extracellular concentrations. Average cell size is consistent at room temperature with two exceptions. When the outside K⁺ is lower than 6.5 mM the cells tend to be smaller with less intracellular K⁺ and high Ca⁺⁺. In stationary phase cultures the cells are larger with high intracellular Mg⁺⁺ and low K⁺.     

Polyol concentrations in Aspergillus repens grown under salt stress

Na⁺, K⁺ and the ratio of Na⁺/K⁺ were higher in cells of the halotolerant Aspergillus repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na⁺/K⁺ ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.     

Response of sugarcane to different types of salt stress

Summary Due to climatic conditions and prevailing water regime the yield and sucrose recovery in sugarcane are high in South Western India. However, excessive irrigation, poor drainage and luxuriant use of fertilizers have resulted in conversion of large fertile areas into saline lands. The salinity is due to the excess of Na⁺, Ca⁺⁺, Mg⁺⁺, SO₄ ^– and Cl^– ions. Individual salts of NaCl, Na₂SO₄, MgCl₂ and MgSO₄ were employed in culture experiments to study salt stress effect on sugarcane variety Co 740. It was observed that sulphate salinity was more toxic to sugarcane than the chloride one. Sulphate salts caused more inhibition of growth, chlorophyll synthesis, PEPCase activity, decreased the uptake of K⁺ and Ca⁺⁺ ions but stimulated nitrate reductase. The stress did not result in proline accumulation in the sugarcane cultivar Co 740. The degree of toxicity of different ions in decreasing order in sugarcane cultivar Co 740 is SO₄ ^–>Na⁺>Cl^–>Mg⁺⁺.     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

Early changes of 'leak flux' and the cation content of lymphocytes by concanavalin A.

The leak fluxes of Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in mouse thymocytes are increased by Concavaline A (Con A), within minutes after mitogen addition. The intracellular Mg⁺⁺ and K⁺ concentrations were decreased and the Na⁺ and Ca⁺⁺ contents were increased by Con A in mouse thymocytes and spleen cells.     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

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.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.