Evidence for a role of Na+,K(+)-ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation.

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K(+)-ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non-hydrating oocytes for Na+,K(+)-ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle-enclosed oocytes in K(+)-free medium or with ouabain or amiloride, inhibitors of Na+,K(+)-ATPase and Na+ channels, respectively, blocked gonadotropin-induced oocyte hydration in M. undulatus. In addition, Na+,K(+)-ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain-sensitive Na+,K(+)-ATPase system in oocyte hydration in two species of sciaenid fishes.     

Effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae cells. Studies by 13C-, 1H-NMR and biochemical methods

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1-13C]glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using 13C-, 1H-NMR and biochemical methods. The 13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 1H-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 microM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The 13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (approximately 1.0) is practically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 microM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.     

Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities.

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.     

Permeability changes in the cytoplasmic membrane of Escherichia coli K-12 early after infection with bacteriophage T1.

The nature of the bacteriophage T1-induced changes in the permeability of the cytoplasmic membrane of Escherichia coli K-12 was investigated. At 20 degrees C and with glucose as a substrate, the addition of one bacteriophage per cell induced a complete and irreversible loss of K+ ions (single-hit phenomenon). K+ loss was compensated by an uptake of Na+, Li+, or choline by the cell, depending on which of these ions was the major cation in the medium. T1 depolarized the cells and inhibited 86Rb+-K+ exchange across the cytoplasmic membrane. The loss of K+ occurred independently of the Mg2+ concentration in the medium. By contrast, at low but not at high Mg2+ concentrations, T1 caused efflux of Mg2+ which in turn caused inhibition of respiration and a decrease of delta pH.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Activities of enzymes from canine kidney plasma membranes under effects of polyene antibiotics in vivo and in vitro]

The effects of amphotericin B drug containing sodium deoxycholate (DOC) and those of DOC and nistatin on the activities of Na+, K+-ATPase and 5'-nucleotidase of canine kidney plasma membranes were studied. It was found that the activities of Na+, K+-ATPase and 5'-nucleotidase were markedly inhibited only after intravenous injection of amphotericin B, whereas the other agents tested caused no changes in the enzyme activities. Similar results were obtained in vitro. In the presence of amphotericin B the activity of Na+, K+-ATPase was noticeably inhibited already at the antibiotic concentration of 0,1 mkg per mg of membrane protein. It was found that the injection of amphotericin B, DOC and nistatin did not qualitatively or quantitatively affect the phospholipid composition of the plasma membranes. This is indicative of the lack of correlation between the enzyme activities and changes in the phospholipid composition of the plasma membranes under effects of amphotericin B. The pyrimidine derivative--amygluracyl--markedly removes the inhibiting effect of amphotericin B on the enzyme activity of plasma membranes.     

Glucose inhibits 45Ca efflux from pancreatic beta-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca2+, addition of glucose or omission of Na+ resulted in prompt and quantitatively similar inhibitions of 45Ca efflux from beta-cell rich pancreatic islets microdissected from ob/ob mice. Glucose had no additional inhibitory effect when Na+ was isoosmotically replaced by sucrose or choline+. When K+ was used as a substitute for Na+, the inhibitory effect of Na+ removal on 45Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting 45Ca efflux in the presence or absence of Na+ is difficult to reconcile with the postulate that the Na+-Ca2+ countertransport mechanism is a primary site of action for glucose.     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Na+ modulates the K+ permeability and the membrane potential of alkalophilic Bacillus

In the absence of Na+ in the medium, the membrane potential of obligately alkalophilic Bacillus cells was found to be decreased by the addition of K+ to the medium, whereas K+ addition in the presence of Na+ had no effect. Rb+ showed essentially the same effect as K+. The decreased membrane potential was quickly restored by lowering the K+ concentration in the medium or by adding Na+ or Li+ to the medium. Thus, in the absence of Na+, the membrane potential of alkalophilic Bacillus seems to be affected by the concentration difference of K+ between inside and outside of the cell, and Na+ or Li+ in the medium suppresses the K+ effect. An exchange between extracellular Rb+ and intracellular K+ was observed in the absence of Na+. However, the exchange was greatly suppressed by the addition of Na+ or Li+ to the medium, indicating that Na+ in the medium modulates the K+ permeability of the alkalophilic Bacillus cell membrane. The K+-induced decrease in the membrane potential of alkalophilic Bacillus in the absence of Na+ is accounted for by the increased K+-permeability of the cell membrane.     

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.     

Structural-functional changes in the synaptic membranes of the cerebral cortex of rats during electric stimulation in vitro]

Electric stimulation (EC) of a suspension of native synaptic membranes of rat brain cortex in the Krebs-Ringer-glucose medium revealed Ca-dependent inhibition of Na+, K+-ATPase and inhibition of transport Ca-activated, Mg-dependent ATPase. The effects observed are not induced by a change in the SH-groups of the membrane proteins and are removed by an addition of total lipids of the brain (membrane protein: lipid = 5:1) or 0.35 mM novocaine. Cyclic 3',5'-AMP in concentrations of 0.1--1.0 mM causes an inhibition (up to 50%) of Na+, K+-ATPase of native synaptic membranes. The Na+, K+-ATPase activity of purified membrane preparations is not changed either by the cyclic nucleotide, or by EC. It is assumed that depolarization of excitable membranes results in structural changes, mediated by the activation of protein kinase, and manifesting themselves as labilization of protein-lipid ratios.     

Effect of heavy metal cations on the activity of membrane Na+, K+ -ATPase in embryos of loach (Misgurnus fossilis L.)

The activity of membrane Na+, K+ -ATPase of embryos of loach (Misgurnus fossilis L.) at early stages of development in the normal conditions and under the influence of heavy metal cations has been investigated. It was established, that the influence of such heavy metal cations as Ni2+, Co2+, Sn2+, Zn2+, Mn2+ and Cd2+ in concentrations 10(-6) - 10(-4) M results in reduction of activity of membrane Na+, K+ -ATPase of loach embryos. It was shown, that the inhibition effect is more expressed with the increase of concentrations of heavy metal cations in the incubation medium. The definition of inhibition constants Io.5 has allowed to analyze the sensitivity of Na+, K+ -ATPase to influence of various cations of heavy metals at different stages of blastomer division. Possible mechanisms of influence of heavy metal cations on the activity of membrane Na+, K+ -ATPase of loach embryos have been considered.     

The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na+, K+)-ATPase.

A rat heart plasma membrane preparation isolated in a sucrose medium and some of its enzymatic properties have been investigated. It has been shown that a rat heart plasma membrane fraction contains high creatine phosphokinase activity which can not be diminished by repeated washing with sucrose solution. Creatine phosphokinase extracted from a plasma membrane fraction with potassium chloride and 0.01% deoxycholate solution is electrophoretically identical to MM isoenzyme of creatine phosphokinase. Under the conditions where (Na+,K+)-ATPase is activated by addition of Na+, K+ and MgATP, creatine phosphokinase of plasma membrane fraction is able to maintain a low ADP concentration in the medium if creatine phosphate is present. The rate of creatine release is dependent upon MgATP concentration in accordance with the kinetic parameters of the (Na+,K+)-ATPase and is significantly inhibited by ouabain (0.5 mM). The rate of creatine release is also dependent on creatine phosphate concentration in conformance with the kinetic parameters of MM isozyme of creatine phosphokinase. It is concluded that in intact heart cells the plasma membrane creatine phosphokinase may ensure effective utilization of creatine phosphate for immediate rephosphorylation of ADP produced in the (Na+,K+)-ATPase reaction.     

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.     

Oscillation of electrical potential in a porous membrane doped with glycerol alpha-monooleate induced by an Na+/K+ concentration gradient

The electrical potential across a fine-pore membrane doped with glycerol alpha-monooleate and separating aqueous solutions of 0.5 M NaCl and 0.5 M KCl was studied. It was found that this system showed rhythmic and sustained oscillations of electrical potential. These oscillations may be due to the phase transition of glycerol alpha-monooleate molecules within the fine pores. In relation to this, it is shown here that Na+ and K+ have different effects on the aggregation of glycerol alpha-monooleate. This oscillatory phenomenon is very interesting because in biological nervous membrane an Na+/K+ concentration difference across the membrane is essential for excitability.     

Ca2(+)-dependent glucose transport in chicken intestine

Transport of glucose was measured in the intestine of white leghorn layers in vivo using ligated upper small intestinal segment in the presence of Ca2+ and other ions either singly or in combination. Transport of glucose across the intestine was very significantly increased with Ca2+ than Na+, K+ and Po4(3-) individually, but when Ca2+ was combined with Na+, K+ and PO4(3-), the glucose absorption increased significantly over that achieved by Na+ ions alone. These data revealed that Ca2+ ions might be exerting the major influence on glucose transport processes of the chicken intestine.     

Osmoadaptation in representatives of haloalkaliphilic bacteria from soda lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K+, Na+, and Cl- ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na+ in the medium were revealed in this organism. At a concentration of 1.91 M Na+ in the medium, N. acetigena accumulated 0.83 M K+, 0.91 M Na+, and 0.29 M Cl- in cells, and, with an increase in the Na+ content in the medium to 2.59 M, it accumulated 0.94 M K+, 1.98 M Na+, and 0.89 M Cl-, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Guanidinium derivatives act as high affinity antagonists of Na+ ions in occlusion sites of Na+,K(+)-ATPase.

We have screened various alkyl- and arylguanidinium derivatives as possible competitors of Na+ or Rb+ for the cation sites on renal Na+,K(+)-ATPase. Alkyl-monoguanidinium or alkylbisguanidinium (BisG) compounds (chain lengths of C3 to C10) competitively inhibit the occlusion of Rb+ and Na+ with an order of affinities C10 greater than C8 greater than C6 greater than C4 greater than C3. BisG compounds are approximately twice as effective as the equivalent alkylmonoguanidinium compounds. In media of high ionic strength, affinities of tens of micromolar are observed, e.g. 26 microM for BisG 8. m-(mXBG)- and p-xylylenebisguanidinium were synthesized and were found to compete with Rb+ or Na+ with intrinsic affinities of 7.7 and 8.2 microM, respectively. The hydrophobicity rather than the degree of proximity of the guanidinium groups in all BisG compounds appears to determine the binding affinity. A systematic search has been made of conditions in occlusion assays for which the inhibitor affinities are highest. When the pH is raised from 7.0 to 8.5, a 5-fold increase in affinity is observed, suggesting that the guanidinium derivatives compete with protons at sites of pKa approximately 7.5. Replacing Tris-HCl with choline chloride-containing media raised apparent affinities approximately 2-fold. All guanidinium derivatives stabilize the E1 conformation of fluorescein-labeled Na+,K(+)-ATPase, acting as competitive Na+ analogues. In media containing only 1 mM Tris-HCl, pH 8.55, very high affinities were observed for binding to the fluorescein-labeled enzyme (e.g. 0.08 microM for mXBG). In very low ionic strength medium, the inhibition was still competitive with Rb+ ions. However, there was also evidence for nonspecific adsorption to the membranes. The following findings show that mXBG, a typical guanidinium derivative, behaves as a Na(+)-like antagonist. (a) It inhibits Na+,K(+)-ATPase activity, competing strongly with Na+ but only weakly with K+ ions. (b) It inhibits phosphorylation from ATP, competing with Na+ ions. (c) Like Na+ ions, it blocks phosphorylation from inorganic phosphate. Based on these results, we propose that the guanidinium group binds to a relatively wide vestibule at the cytoplasmic surface; but, unlike Na+ or K+ ions, it cannot pass into a narrower region of the cation transport path within the membrane. Therefore, it blocks the occlusion and active transport of cations. In the future, high affinity guanidinium derivatives may serve the purpose of locating cation-binding domains of the pump protein after being converted to reactive affinity or photoaffinity covalent labels.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies.

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.