The effects of mineral nutrition and benzyladenine on the plasmalemma ATPase activity from roots of wheat and Plantago major ssp. pleiosperma

There is an increasing awareness of the possibilities of mineral nutrition as regulator of growth substance action and vice versa. The present paper focuses on the effects of mineral nutrition and benzyladenine at the level of the plasma membrane. Seedlings of wheat ( Triticum aestivum L. cv. Drabant) and juvenile plants of Plantago major L. ssp. pleiosperma (Pilger) were grown hydroponically at different mineral levels with or without benzyladenine. Purified plasmalemma preparations from roots of wheat and P. major ssp. pleiosperma were obtained by the two phase partitioning method, using 6.5% (w/w) of each of Dextran T-500 and polyethylene glycol 3350. The Mg²⁺ and (Mg²⁺+ K⁺) dependent ATPase activities of the root plasmalemma in both species and the (Mg²⁺+ Cl⁻) one in P. major ssp. pleiosperma increased with increasing mineral levels, but the ionic strength did not influence the substrate specificity, the sensitivity to inhibitors or the pH optima.
The addition of 10⁻⁸ M benzyladenine to a nutrient solution increased the ATPase activities. The pH optima and the sensitivity to several inhibitors were not affected by benzyladenine, but the substrate specificity for ATP decreased, except for the K⁺ stimulation. In conclusion, benzyladenine mimics the effects of a higher mineral level than actually applied. Data from this and previous experiments indicate that benzyladenine exerts its effects by increasing the endogenous cytokinin concentrations and by modulating membrane components.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

The surface charge density of wheat root membranes

Seedlings of winter wheat ( Triticum aestivum L. cv. Hildur) were grown at 18°C for 7 days in darkness in a complete growth medium in the presence or absence of 1 m M KCl to produce roots with different ion contents (high and low K⁺ respectively). The roots were homogenized, the 3 000 g, 10 000 g, 30 000 g (further fractionated by two phase partitioning) and 100 000 g pellets isolated, and their surface charge densities (σ) determined by the use of 9-aminoacridine fluorescence. The average σ for all membrane fractions weighted for protein content was the same (−18 mC m⁻²) for low and high K⁺ roots. The K⁺, Na⁺, Mg²⁺ and Ca²⁺ content of roots was determined and used to calculate an average σ following the procedure of Bérczi et al. [Physiol. Plant. 61: 529–534 (1984)]. The predicted value (−11 mC m⁻²) does not deviate much from the experimentally determined value. It is concluded as a useful working hypothesis that the average surface charge density is constant and that the ionic content of plant cells is regulated such that the average surface potential is constant.     

Relation of source and sink during grain filling period in wheat and some aspects of its regulation

The effect of Mg²⁺, Na⁺, K⁺, ouabain and pH on ATPase activity of purified membrane fractions enriched in plasmalemma fragments from Hordeum vulgare L. (glycophyte) and Halocnemum strobilaceum L. (halophyte) was studied. Membrane ATPases from both plants were synergistically activated by K⁺ and Na⁺ in the presence of Mg²⁺. The maximum activity of the enzymes were observed at the ratio Na/K = 2–3. Ouabain (10^-4 M) almost completely eliminated the (Na⁺+ K⁺)-stimulated component of the ATPase activity. The Na, K, Mg-ATPase of Hordeum had a single pH optimum (pH 8), but that of the Halocnemum had two optima(pH 6 and 8). It appears that similar enzymes operate in the cells of both plants studied. The higher Na, K, Mg-ATPase activity of the halophyte compared to that of the glycophyte suggests the involvement of the enzyme in the extrusion of Na⁺ from the cytoplasm of cells of both plants.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Distribution of ATPases in wheat root membranes separated by phase partition

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots ( Tritium aestivum L . cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g , 10,000 g , 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two-phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter-current distribution id a 6.3/6.3% two-phase system. Protein and activities of Ca²⁺, Mg²⁺, and Mn²⁺ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca²⁺, Mg²⁺ or Mn²⁺ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3-system was selected for further separation of the membranes in the 30 KP fraction by counter-current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.     

Nisin, temperature and pH effects on growth and viability of Pectinatus frisingensis, a Gram-negative, strictly anaerobic beer-spoilage bacterium

Pectinatus frisingensis , a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin. An increase in nisin concentration (0 to 1100 IU ml⁻¹) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium. In addition, late exponential cells of P. frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K⁺. Presence of Mg²⁺ up to 15 mmol l⁻¹ did not protect P. frisingensis from nisin-induced loss of viability and K⁺ efflux. Potassium leaks were also measured in P. frisingensis late exponential phase cells exposed to combined effects of nisin addition (100–500 IU ml⁻¹), 10 min mild heat-treatment (50 °C) or rapid cooling (2 °C), and pH (4·0 and 6·2). Net K⁺ efflux from both starving and glucose-metabolizing cells, was more important at pH 6·2, whatever the temperature treatment and nisin addition. Reincubation at 30 °C of P. frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K₊ than heat-treated cells, whatever the pH conditions. A combination of nisin and mild heat-treatment could thus be of interest to prevent P. frisingensis growth in beers.     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Modulation of mechanosensitive calcium-selective cation channels by temperature

Gating of associations of mechanosensitive Ca²⁺-selective cation co-channels in the plasmalemma of onion epidermis has a strong and unusual temperature dependence. Tension-dependent activity rises steeply as temperature is lowered from 25°C to about 6°C, but drops to a low level at about 5°C. Under the conditions tested (with Mg²⁺ and K⁺ at the cytosolic face of outside-out membrane patches), promotion results both from more bursting at all observed linkage levels and from longer duration of bursts of co-channels linked as quadruplets and quintuplets. Co-channel conductance decreases linearly, but only modestly, with declining temperature. It is proposed that these and related mechanosensitive channels may participate in a variety of responses to temperature, including thermonasty, thermotropism, hydrotropism, and both cold damage and cold acclimation.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Membrane and ion transport properties in cereals under acidic and alkaline stress

The effects of pH on the growth and the K⁺ (⁸⁶Rb) uptake and K⁺ content of excised rice ( Oryza sativa L. cv. Dunghan Shali) and wheat ( Triticum aestivum L. cv. GK Szeged) roots were investigated. Rice roots responded to H⁺ stress with an increased K⁺(⁸⁶Rb) influx and a decreased K⁺ content, suggesting an increased exchange between the cytoplasmic K⁺ pool and the external medium. Under the same experimental conditions wheat did not show any anomalous K⁺(⁸⁶Rb) influx. Growth of both rice and wheat was relatively insensitive to pH between 4 to 10.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Uptake and distribution of calcium, magnesium and potassium in cucumber of different age

Uptake and distribution of Ca⁺, Mg²⁺ and K²⁺ were investigated in plants of cucumber ( Cucumis sativus L. var. Cila) which had been cultivated for 12, 19, 32, or 53 days in complete nutrient solution with 1.0 m M Ca²⁺, 2.0 m M Mg²⁺ and 2.0 m M K⁺. The ⁺ concentration was about the same in roots and shoots, while the Ca²⁺ and Mg²⁺ concentrations were low in roots compared to shoots. The K⁺ concentration decreased with increasing leaf age, while the Ca²⁺ and Mg²⁺ concentrations increased, except in older plants with flowers and fruits, where an increased concentration was found in the youngest leaves. This is discussed in connection with increased indoleacetic acid (IAA) synthesis in the shoot. Excision of leaves at different levels from 21-day-old plants, followed by uptake for 24 h from the nutrient solution on days 22 and 23, resulted in no immediate reduction in Ca²⁺ (⁴⁵Ca) uptake. Transport of Ca²⁺ increased to leaves above and below the excision point and total Ca²⁺ uptake remained at the same level as for the intact plant. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root while the distribution in the shoot is regulated by the accessability of negative binding sites.     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.