Synthesis and evaluation of panaxatriol derivatives as Na+, K+-ATPase inhibitors

Panaxatriol, a triterpene bearing a steroid-like structure similar to cardiac glycosides, was presumed to share the same bioactivity with cardiac glycosides, and may be a potential Na⁺, K⁺-ATPase inhibitor. In this paper, a series of panaxatriol derivatives were synthesized and evaluated for Na⁺, K⁺-ATPase inhibitory activities. The results of biological tests showed that more than half of the synthesized derivatives presented increased inhibitory activities compared with panaxatriol. Of these compounds, 13a with a 3, 4-seco skeleton showed the most potent inhibitory activity, which was equal to that of the standard drug digoxin. To understand the binding mode of the most active compound, molecular docking study of 13a with Na⁺, K⁺-ATPase was conducted. Therefore, 13a may serve as a new lead compound for the development of novel Na⁺, K⁺-ATPase inhibitors.     

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers,leaves, and twigs of Streblus asper

A new non-cytotoxic [(+)-17β-hydroxystrebloside (1)] and two known cytotoxic [(+)-3′-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na⁺/K⁺-ATPase. Compound 3 docks deeply in the Na⁺/K⁺-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4′ hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na⁺/K⁺-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na⁺/K⁺-ATPase. Thus, 3 was found to inhibit Na⁺/K⁺-ATPase, but 1 did not. In addition, the cytotoxic and Na⁺/K⁺-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na⁺/K⁺-ATPase but not by interacting with glucose transporters.     

Endogenous Digitalis-Like Na+, K+-ATPase Inhibitors,and Brain Function

Digitalis-like compounds are recently identified steroids synthesized by the adrenal gland, which resemble the structure of plant cardiac glycosides. These compounds, like the plant steroids, bind to and inhibit the activity of the Na⁺, K⁺-ATPase. The possible function of the endogenous digitalis-like compounds has to be evaluated in view of the presence of different isoforms of the Na⁺, K⁺-ATPase, which differ in their sensitivity to digitalis. This review focuses on recent published data on the Na⁺, K⁺-ATPase inhibitors, the digitalis-like compounds, regarding their structure, biosynthesis and secretion from the adrenal gland, physiological role and pathological implications in diseases such as hypertension and depression. Emphasis is given to studies describing the involvement of these compounds in brain function.     

Transport ATPase of erythrocyte membrane: sensitivities of Na plus, K, plus-ATPase and Kplus-phosphatase activities to ouabain.

Cardiac glycosides are inhibitors of Na⁺,K⁺-ATPase, and K⁺-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K⁺-phosphatase is significantly less sensitive than Na⁺,K⁺-ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methyl-fluorescein phosphate was used as substrate for the continuous fluorimetric assay of K⁺-phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K⁺-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K⁺ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na⁺,K⁺-ATPase and K⁺-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na⁺,K⁺-ATPase and K⁺-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

Effects of vanadate on Na+,K+-ATPase and on the force of contraction in guinea-pig hearts.

Vanadate has been shown to be a potent inhibitor of isolated Na⁺,K⁺-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na⁺,K⁺-ATPase. Vanadate inhibited isolated Na⁺,K⁺-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K⁺-stimulated phosphatase activity was more sensitive than Na⁺,K⁺-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [³H] oubain binding in the presence of Mg²⁺, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive ⁸⁶Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na⁺,K⁺-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na⁺,K⁺-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

Digitalis receptor sugar binding site characteristics: A model based upon studies of Na+, K+-ATPase preparations with differing digitalis sensitivities

The structure-activity relationships of the genin moieties of digitalis glycosides are commonly elucidated by determining the inhibitory potency of a variety of genins toward the plasma membrane Na⁺, K⁺-ATPase; qualitatively these relationships appear to be fairly independent of the specific Na⁺, K⁺-ATPase preparation utilized for the analysis. To determine whether this is the case with regard to the sugar moieties of glycosides, the inhibitory effects of 12 monoglycosides of digitoxigenin toward four Na⁺, K⁺-ATPase preparations of different origin were measured. It was found that while recognition of the major structural determinants of sugar activity appeared to be independent of enzyme source, recognition of the minor structural determinants of activity showed some source dependence. It was also observed that the intrinsic sensitivity to sugar potentiation may be source dependent and unrelated to intrinsic sensitivity to inhibition by digitoxigenin. These observations are compatible with a model of the Na⁺, K⁺-ATPase sugar binding site(s) in which intrinsic sensitivity to sugar attachment as well as recognition characteristics (for sugar structural features) both determine the extent to which a sugar moiety may contribute to the activity of monoglycosides. Further, in these studies one of the Na⁺, K⁺-ATPase preparations employed was obtained from rat brain, a tissue known to contain a mixture of ouabain sensitive and insensitive isoforms. We have observed that the rigorous purification techniques employed appear to have selectively removed from or denatured the less ouabain sensitive al isoform found in this enzyme preparation.     

LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH : I. Biochemical Characterization of Subcellular Fractions

In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na⁺, K⁺-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na⁺, K⁺-ATPase and ouabain insensitive Mg²⁺-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg²⁺-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na⁺, K⁺-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na⁺, K⁺-ATPase in M+L appeared to be associated with structures containing no Mg²⁺-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na⁺, K⁺-ATPase activity was highly dependent on the ratio of Na⁺ and K⁺ concentrations but independent of absolute concentrations over at least a fourfold range.     

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na⁺, K⁺)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 ‰ salinity) or acclimated to 21 ‰S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na⁺, K⁺)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na⁺, K⁺)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a V_max of 54.9 ± 1.8 nmol min^?1 mg^?1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg²⁺ affinity increased with salinity acclimation while K⁺ affinity decreased. (Ca²⁺, Mg²⁺)-ATPase activity increased while V(H⁺)- and Na⁺- or K⁺-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na⁺, K⁺)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na⁺, K⁺)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.     

The Decrease on Na+, K+-ATPase Activity in the Cortex, but not in Hippocampus, is Reverted by Antioxidants in an Animal Model of Sepsis

In the present study, we investigated whether sepsis induced by cecal ligation and puncture (CLP) modifies Na⁺, K⁺-ATPase activity, mRNA expression, and cerebral edema in hippocampus and cerebral cortex of rats and if antioxidant (ATX) treatment prevented the alterations induced by sepsis. Rats were subjected to CLP and were divided into three groups: sham; CLP??rats were subjected to CLP without any further treatment; and ATX?CCLP plus administration of N-acetylcysteine plus deferoxamine. Several times (6, 12, and 24) after CLP or sham operation, the rats were killed and hippocampus and cerebral cortex were isolated. Na⁺, K⁺-ATPase activity was inhibited in the hippocampus 24?h after sepsis, and ATX treatment was not able to prevent this inhibition. The Na⁺, K⁺-ATPase activity also was inhibited in cerebral cortex 6, 12, and 24?h after sepsis. No differences on Na⁺, K⁺-ATPase catalytic subunit mRNA levels were found in the hippocampus and cerebral cortex after sepsis. ATX treatment prevents Na⁺, K⁺-ATPase inhibition only in the cerebral cortex. Na⁺, K⁺-ATPase inhibition was not associated to increase brain water content. In conclusion, the present study demonstrated that sepsis induced by CLP inhibits Na⁺, K⁺-ATPase activity in a mechanism dependent on oxidative stress, but this is not associated to increase brain water content.     

Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.