Greater sensitivity to vanadate of rat renal relative to cardiac (Na++K+)-ATPase

Vanadate (VO₄^?3) produces a positive inotropic effect in rats and also promotes diuresis as well as natriuresis. Although the mechanism(s) of these effects is uncertain, in the kidney, VO₄^?3 may act through inhibition of (Na⁺+K⁺)-ATPase activity, whereas in the heart, other or additional mechanisms are likely. Under the assay conditions used in the present study, microsomal (Na⁺+K⁺)-ATPase activities from rat kidney cortex and medulla were inhibited to a greater extent than was left ventricular (Na⁺+K⁺)-ATPase activity over a range of VO₄^?3 concentrations. The apparent dissociation constant for left ventricular (Na⁺+K⁺)-ATPase (10.95 ± 1.26 × 10^?7M VO₄^?3) was significantly greater than that of (Na⁺+K⁺)-ATPase from the cortex (3.46±0.96×10^?7 M VO₄^?3) or the medulla (3.32±0.7×10^?7M VO₄^?3, N=6, P<.05) whereas there were no significant differences between the effects of VO₄^?3 on (Na⁺+K⁺)-ATPase from the cortex and medulla. The greater inhibition by VO₄, of (Na⁺+K⁺)-ATPase from the cortex relative to that of the left ventricle, occurred over a range of Na⁺ and K⁺ concentrations, and K⁺ enhanced the inhibition by VO₄^?3 to a greater extent for (Na⁺+K⁺)-ATPase from the cortex than the left ventricle. These results suggest that renal (Na⁺+K⁺)-ATPase is more sensitive than left ventricular (Na⁺+K⁺)-ATPase to inhibition by VO₄^?3 and would, therefore, be more likely to be modulated $\text{in}$$\text{vivo.}$     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

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

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

Effect of reduced pH in absence of HCO3− on anomalous and normal potential responses in bullfrog antrum

Effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO₃^? but with 95% O₂/5% CO₂ in both cases. In both cases, changing from 4 to 40 mM K⁺ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO₃^?. Changing from 102 to 8 mM Na⁺ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO₃^? was insignificant but with 25 mM HCO₃^? the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K⁺ and Na⁺ were largely related to ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump. Changes in nutrient Cl^? from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pump and Na⁺/Cl^? symport. Removal of HCO₃^? and concurrent reduction of pH modify resistance of these pathways.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation

Frozen aqueous suspensions of partially purified membrane-bound renal (Na⁺ + K⁺)-ATPase have been irradiated at –135°C with high-energy electrons. (Na⁺ + K⁺)-ATPase and K⁺-phosphatase activities are inactivated exponentially with apparent target sizes of $\text{184 ± 4 kDa and 125 ± 3 kDa}$, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na⁺ + K⁺)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na⁺?K⁺ exchange, $\text{201 ± 4}\text{kDa}$; (ATP + P_i)-activated Rb⁺?Rb⁺ exchange, $\text{206 ± 7}\text{kDa}$ and ATP-independent Rb⁺?Rb⁺ exchange, $\text{117 ± 4}\text{kDa}$. The apparent size of the α-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the β-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na⁺ + K⁺)-ATPase is αβ. (2) The inactivation target size for (Na⁺ + K⁺)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na⁺ and K⁺. (3) The target sizes, for K⁺-phosphatase (125 kDa) and ATP-independent Rb⁺?Rb⁺ exchange (117 kDa) are indistinguishable from that of the α-chain itself, suggesting that cation binding sites and transport pathways, and the $\text{p-}\text{nitrophenyl}$ phosphate binding site are located exclusively on the α-chain. (4) ATP-dependent activities appear to depend on the integrity of an αβ complex.     

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K⁺ on Na_o⁺-dependent Ca²⁺ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K⁺ (up to 70 mM) has no effect on Na⁺?Ca²⁺ exchange. Removal of K_i⁺ causes a marked inhibition in the Na_o⁺-dependent Ca²⁺ efflux component. Internal K⁺ activates the Na⁺?Ca²⁺ exchange with low affinity $\text{(K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 90 mM)}$. Activation by K_i⁺ is similar in the presence or in the absence of Na_i⁺, thus ruling out a displacement of Na_i⁺ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca²⁺ efflux on K_i⁺. The present results demonstrate that K_i⁺ is an important cofactor (partially required) for the proper functioning of the forward Na⁺?Ca²⁺ exchange.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Induction of passive monovalent cation-exchange activity in heart mitochondria by depletion of endogenous divalent cations

Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive ${}^{42}\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K⁺ (K_m = 25 mm). Intramitochondrial ⁴²K⁺ in cation-depleted mitochondria exchanges passively with external Na⁺ and Li⁺, but not with choline⁺. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient.     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.     

Influence of ouabain on Na+ and K+ concentration in haemolymph of Drosophila hydei and appearance of Malpighian tubules

After injection of ouabain into the body cavity, the Malpighian tubules of Drosophila larvae show a characteristic appearance which differs from the normal. The primary urine, which is mainly found in particles, is diluted and the concretions are washed away through the proximal part of the tubule and the ureter into the hindgut. In the haemolymph the Na⁺ and K⁺ concentrations change significantly. The K⁺ concentration increases rapidly to double the normal, while later the Na⁺ concentration rises up to 2·3 times the normal. Water movements are not the cause of the concentration changes because the quotient $\text{Na}\text{K}$ varies widely. Thus primary ion regulation mechanisms are influenced in the insect body by application of g-strophanthin. This is evidence for the existence of a ouabain-sensitive ATPase, which is decisively involved in the ion transport mechanisms in the insect body.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabian-insensitive fluxes of thallium ion in erythrocytes

A maximal rate of the ouabain-sensitive ²⁰⁴Tl influx in human erythrocytes can be attained at trace concentrations of Tl⁺ in Mg²⁺ isotonic media free of K⁺ and Na⁺. The maximal influx of Tl⁺ from isotonic Mg(NO₃)₂ at 20°C and pH 7.4 was $\text{0.45}\text{mM}\text{· 1}{}^{\mathrm{?1}}\text{· h}{}^{\mathrm{?1}}$ with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.025 mM. In contrast to the active influx of Tl⁺, the passive Tl⁺ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl⁺ and K⁺ studied. The rate constants of Tl⁺ passive fluxes in human and cat erythrocytes can be related to pH by the equation $\text{log}\text{k}{}_{\mathrm{<mtext>in(out)</mtext>}}\text{= –A + B ·}\text{pH}$, where $\text{A}$ and $\text{B}$ are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl⁺ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl⁺ is about two orders of magnitude faster than those of K⁺ or Rb⁺. An extra non-Coulombic interaction between Tl⁺ and membrane ligands appears to be involved providing an accumulation of Tl⁺ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl⁺ in both directions.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K⁺-dependent phosphatase reaction. K⁺ activation and Na⁺ inhibition of this reaction are described quantitatively by a model featuring isomerization between E₁ and E₂ enzyme conformations with activity proportional to E₂K concentration:

Differences between the three preparations in $\text{K}{}_{0.5}$ for K⁺ activation can then be accounted for by differences in equilibria between E₁K and E₂K with dissociation constants identical. Similarly, reductions in $\text{K}{}_{0.5}$ produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E₂ conformations. Na⁺ stimulation of K⁺-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E₁Na but dependent also on K⁺ activation through high-affinity sites. With inside-out red blood cell vesicles, K⁺ activation in the absence of Na⁺ is mediated through sites oriented toward the cytoplasm, while in the presence of Na⁺ high-affinity K⁺-sites are oriented extracellularly, as are those of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase reaction. Dimethyl sulfoxide accentuated Na⁺-stimulated K⁺-dependent phosphatase activity in all three preparations, attributable to shifts from the E₁P to E₂P conformation, with the latter bearing the high-affinity, extracellularly oriented K⁺-sites of the Na⁺-stimulated pathway.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.