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.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

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.     

Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium

Light-induced Na⁺ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na⁺ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na⁺ efflux, the inhibition being complete at low light intensity. The Na⁺ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na⁺ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na⁺ transport. Proton influx in the dark followed the artificial formation of a sodium gradient ($\text{[Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{in}}\text{> [Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{out}}$). These results may be explained by a Na⁺/H⁺ antiport mechanism. The fluxes of Na⁺ and H⁺ were of comparable magnitude, but the initial rate of Cl^? efflux in the same experiment was one-third of the initial rate of Na⁺ efflux. Consequently Cl^? is not regarded as a participant in the Na⁺ efflux mechanism.     

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 μg per calf bladder. Low levels of 5′ nucleotidase, Mg²⁺-ATPase and ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000 and 22 000. These may associate to form a 30 000 apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ complex which could represent the individual ‘particles’ of the dodecameric subunits seen by electron microscopy in the plaque regions.     

Catecholamine-stimulation of Cl− secretion in MDCK cell epithelium

Cultured epithelial monolayers of MDCK cells grown upon Millipore filter supports and mounted in Ussing chambers for transport studies respond to addition of $\text{5 · 10}{}^{\mathrm{?7}}\text{M}$ adrenalin from only the basal bathing solution by an increased short-circuit current, due both to an increased transmonolayer potential difference (basal solution electropositive) and an increased transmonolayer conductance. Measurement of tracer Na⁺, K⁺ and Cl^? fluxes demonstrate that the adrenalin-stimulated short-circuit current results primarily from basal to apical net Cl^? secretion. Half-maximal stimulation of the short-circuit current was observed at ($\text{3.1 ± 0.3) · 10}{}^{\mathrm{?8}}\text{M adrenalin}$; the order of potency of adrenergic agonists for short-circuit current stimulation was $\text{isoprenalin}\text{>}\text{adrenalin}\text{>}\text{noradrenalin}$, consistent with adrenalin action being mediated by a β-adrenergic receptor. The adrenalin-stimulated short-circuit current was sensitive to inhibition (75%) by basal additions of furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$); phloretin inhibition (54%, 57%) was observed from both epithelial surfaces. Amiloride (10^?4 M) and 4-acetamido-4-isothiocyanostilbene-2, 2′-disulphonic acid (SITS) (10 μM) were ineffective as inhibitors of the adrenalin response. The increased short-circuit current was sensitive to replacement of medium Na⁺ by choline (87%) and Tris (93%). Li⁺ was a partially effective substitute cation for Na⁺ · NO₃^?, and isethionate were ineffective substitutes for Cl^? whereas Br^? was partially effective. Partial replacement of medium Na⁺ by choline gave an upward-curving non-saturable dependence of the adrenalin-stimulated short-circuit current upon [Na]; partial replacement of Cl^? by NO₃^? in contrast gave a saturable increase with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approx. 65 mM Cl^?.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

The distribution of (Na++K+)-ATPase along the villus crypt-axis in the rabbit small intestine

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na⁺-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of around 10^?7 M. Both enzyme assay and ouabain-binding measurements suggest that a 2–3-fold increase in the number of Na⁺-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.     

Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen

We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na⁺ channels to localize amiloride-sensitive Na⁺ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M_r's of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M_r's of 2 of the subunits of the renal high amiloride-affintiy Na⁺ channel, namely the and the (=amiloride binding) subunits. The cellular distribution of Na⁺ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were nagative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na⁺ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na⁺ absorption.     

l-Glutamate effects on electrical potentials of synaptic plasma membrane vesicles

The electrogenic nature of the l-glutamate-stimulated Na⁺ flux was examined by measuring the distribution of the lipophilic anion [³⁵S]thiocyanate (SCN^?) into synaptic membrane vesicles that were incubated in a NaCl medium. Concentrations of l-glutamate from 10^?7 to 10^?4 M added to the incubation medium caused an enhanced intravesicular accumulation of SCN^?. Based on the SCN^? distribution in synaptic membrane vesicles it was calculated that 10 μM l-glutamate induced an average change in the membrane potential of + 13 mV. l-Glutamate enhanced both the Na⁺ and K⁺ conductance of these membranes as determined by increases in SCN^? influx. Other neuroexcitatory amino acids and amino acid analogs (d-glutamate, l-aspartate, l-cysteine sulfinate, kainate, ibotenate, quisqualate, $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{aspartate}$, and dl-homocysteate) also increased SCN^? accumulation in synaptic membrane vesicles. These observations are indicative of the activation by l-glutamate and some of its analogs of excitatory amino acid receptor ion channel complexes in synaptic membranes.     

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.     

Harmaline distribution in single muscle fibres and the inhibition of sodium efflux

Harmaline, a known inhibitor of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in cell membranes, inhibited 50% of the ²²Na efflux from barnacle muscle fibres at an extracellular concentration of 2.4 mM. Injected harmaline inhibited 50% of the efflux at an estimated intracellular concentration of about $\text{8}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$, assuming complete equilibration with no binding. Total fibre harmaline was measured in separate fibres by ultraviolet spectrophotometry. Fibres in 3 mM harmaline saline accumulated harmaline with a half-time of 17 min and a final total fibre concentration of $\text{6–12}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$. In harmaline-free saline this accumulated harmaline was lost exponentially with a half-time of 35 min; injected harmaline was lost exponentially from fibres with a half-time of 50 min. It is proposed that harmaline crosses the fibre membrane as the uncharged base and that its apparent accumulation against a concentration gradient is mainly due to intracellular binding with an additional contribution from a transmembrane pH gradient. It is concluded that, in fibres exposed to harmaline saline, the intracellular concentration can reach a sufficiently high value, as judged from the results of the injection experiments, to inhibit Na⁺ efflux at an interior-facing site on the fibre membrane. In contrast, harmaline appears to inhibit the Na⁺-dependent uptake of l-glutamate at an extracellular site.     

Mechanism of action of cesalin, an antitumor protein.

A protein, cesalin, isolated from $\text{Caesalpinia}$$\text{gilliesii}$ is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na⁺, K⁺-ATPase (ATP phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na⁺, K⁺-ATPase. The Na⁺, K⁺-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5′-Nucleotidase and Mg⁺⁺-ATPase are not inhibited by cesalin in any cells tested.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.     

A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes

In genetically low K⁺ but not in high K⁺ red cells of sheep and goat N-ethylmaleimide induced a ouabain insensitive K⁺ flux as measured by tracer influx or net efflux methods. The augmented K⁺ flux was observed in Cl^? or Br^? but not in NO₃^?, SO₄^2? or PO₄^2? media. The action of N-ethylmaleimide was distinct from that of parachloromercuribenzoate or its sulfonic acid derivative which increased both passive K⁺ and Na⁺ movements across the red cell membrane. The instantaneous selective action of N-ethylmaleimide suggests that sulfhydryl groups control a $\text{K}{}^{\mathrm{+}}\text{Cl}{}^{\mathrm{?}}$ transport system which, associated with the low K⁺ gene, is apparently functionally silent in adult ruminant red cells.     

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.     

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.