Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

A possible mechanism for the Na,K-ATPase

A model previously described for the Ca²⁺ pump of sarcoplasmic reticulum has been modified in a thought experiment so that it has the properties of a Na,K-adenosinetriphosphatase (ATPase). When the two Ca²⁺-specific sites are changed into three Na⁺-specific sites, and the channel which opens in the actively transporting conformation made univalent- instead of divalent-cation-selective, the model has the properties of the Na-ATPase which is observed on red cell membranes in the absence of both Na⁺ and K⁺ externally. As in the model for the Ca-ATPase the driving force for transport is generated by a change in solvent structure so that a preformed ionic equilibrium is displaced in favour of less-highly hydrated species; in this case highly hydrated Mg²⁺ ions displace the less highly hydrated Na⁺ ions from binding sites; and Na⁺ diffuses out through a simultaneously opened channel. With the addition of three external K⁺-selective sites per α-polypeptide chain, and the constraint that pump units with their external sites occupied by any univalent cation cannot be phosphorylated by ATP, the model turns out to have the properties of a Na,K-ATPase. It operates in the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, $\text{Na}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ exchange, $\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, K⁺-dependent phosphatase, uncoupled Na⁺ efflux and pump reversal modes. It is concluded that if the modified water in the cleft of the phospho-enzymes has properties similar to those of water at 5°C the pump is competent to exchange three intracellular Na⁺ ions for two extracellular K⁺ ions, and one intracellular Na⁺ ion but it is incapable of exchanging three Na⁺ ions for three K⁺ ions.     

Transductive coupling in bioluminescence: effects of monovalent cations and ionophores on the calcium-triggered luminescence of Renilla lumisomes.

$\text{Renilla}$ lumisomes produce a bioluminescent flash when the vesicles are disrupted with hypotonic solutions containing Ca²⁺. A flash is also observed in the presence of Ca²⁺ using isotonic solutions of monovalent cations under the following conditions: When the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ratio inside the lumisomal membrane is high and when this ratio outside the membrane is low. We suggest that Na⁺ may be the counter ion for Ca²⁺ transport. Na⁺, when outside the membrane, inhibits Ca²⁺-triggered luminescence suggesting that Na⁺ blocks Ca²⁺ channels. Ca²⁺ uptake into the lumisomal membrane, as measured by bioluminescence, is very rapid in the presence of the ionophore A23187. X537A is much less effective. The Ca²⁺ triggered bioluminescence flash observed with lumisomes provides a rapid and sensitive assay for ionophores that are specific for divalent cations such as Ca²⁺.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

The dimerization of ferrihaems: I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization

The dimerization of deuteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (R_obs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, R_obs > 2, whereas for solutions containing mainly dimeric species R_obs < 1.A computer programme has been applied to determine values of the dimerization constant, K, defined as: $\text{K =}\text{[}\text{dimer}\text{][}\text{H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{]}{}^2$.Phosphate buffer anions and Tris · HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order $\text{K}{}^{\mathrm{+}}\text{< Na}{}^{\mathrm{+}}\text{< Li}{}^{\mathrm{+}}\text{<}\text{Sr}{}^{\mathrm{2+}}\text{< Mg}{}^{\mathrm{2+}}\text{? Ca}{}^{\mathrm{2+}}$. Values of K were determined for several concentrations of Na⁺ and Sr²⁺. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation ‘sandwiched’ between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na⁺. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na⁺. The extent of transport inhibition by Na⁺ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na⁺ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values for Na⁺ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order $\text{Li}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Rb}{}^{\mathrm{+}}\text{> Cs}{}^{\mathrm{+}}$. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na⁺, in addition to the competitive inhibition of transport Na⁺ induces an increase in membrane permeability for amino acids.     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca²⁺-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg²⁺ as the only cation) ATPase activity, with virtual elimination of the K⁺-stimulated component. Such treatment causes unmaksing of a latent Mg²⁺-dependent Ca²⁺-stimulated ATPase. Other divalent cations such as Sr²⁺, Ba²⁺, Zn²⁺ and Mn²⁺ were found ineffective as a substitute for Ca²⁺. Moreover, those divalent cations acted as inhibitors of the Ca²⁺-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 70 μM for ATP and the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ values for Mg²⁺ and Ca²⁺ are about $\text{4 · 10}{}^{\mathrm{?4}}\text{M}$ and 10^?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H⁺ transport have been discussed.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Temperature effects on cation affinities of the (Na+, K+)-ATPase of mammalian brain

Effects of temperature on the Na⁺-dependent ADP-ATP exchange and the $\text{p-}\text{nitrophenylphosphatase}$ reactions catalysed by (Na⁺, K⁺)-ATPase were examined. Apparent Mg²⁺ affinity decreased with decreasing temperature. Arrhenius plots of $\text{p-}\text{nitrophenylphosphatase}$ in the presence of Na⁺ and ATP had discontinuities similar to those previously reported for ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, while those of $\text{p-}\text{nitrophenylphosphatase}$ measured without Na⁺ or ATP did not. The apparent activation energy for $\text{p-}\text{nitrophenylphosphatase}$ was a function of the physical characteristics of the cation acting at the K⁺ site.     

Sodium-dependent transport of 5-hydroxytryptamine (serotonin) by canine blood platelets

The transport of 5-hydroxytryptamine (5-HT) was shown to be strongly dependent on the presence of Na⁺ in the incubation medium whereas divalent cations were without effect. The K_m for the Na⁺ requirement was 16.8 mm. The addition of Na⁺ to Na⁺-depleted platelets restored maximum 5-HT transport within 3 min. The affinity of the 5-HT carrier for its substrate was directly proportional to the concentration of Na⁺; however, below 25 mm Na⁺ unique reversible morphological changes in platelet shape occurred as revealed by scanning electron microscopy which resulted in a drastically reduced affinity for 5-HT. K⁺, choline (Ch⁺), or Li⁺ could be used as counterbalancing cations to maintain osmolarity, and the affinity for 5-HT was also dependent on the concentrations of these ions. Ouabain as well as various ionophores at low concentrations inhibited 5-HT uptake. The inhibition was the result of the destruction of the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ gradient across the cytoplasmic membrane. Ionophores, however, did not cause the depletion of either intracellular ATP or 5-HT.     

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.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na⁺ and K⁺ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl₂ and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na⁺ and K⁺ without significantly altering the ratio of pumped $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between ?10 and +60 mV at constant internal pH did not affect the rates of active transport of Na⁺ or K⁺.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.