Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Modification of (Na+,K+)-ATPase function by purified antibodies to the holoenzyme. Effects on enzyme activity and (3H)ouabain binding.

Antibodies (abys) raised to (Na+,K+)-ATPase were purified by elution methods and shown to be cross-reactive with anti-gamma-globulin and the original antigen. Abys were isolated from two different antisera and the effects on (Na+,K+)-ATPase hydrolytic activity and [3H]ouabain binding were measured. The antisera fractions differed as to their maximum level of inhibition of hydrolytic activity and maximal [3H]ouabain binding, but both fractions caused inhibition of maximal [3H]ouabain binding to the same quantitative extent as inhibition of hydrolytic activity. Variable effects on the rate of [3H]ouabain binding were noted which were highly dependent on ligand conditions. During the "turnover state conditions" of the enzyme, the abys stimulated the rate of [3H]ouabain binding to the (Na+,K+)-ATPase. We conclude that effects of aby-(Na+,K+)-ATPase interaction depend upon degree of purity of aby, specificity, aby/enzyme ratios, and ligand conditions.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase.

The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

Modification of the Rate of Ouabain Binding to (Na++ K+)ATPase by Lithium Ions

We report on the interactions of Li+, a congener of K+ with the (Na+ + K+)-ATPase from E Electricus as measured by their effects on the rate of [3H]-ouabain binding to this enzyme. Like K+, Li+ slows ouabain binding under both Type I (Na+ + ATP) and Type II (P1) conditions, but with lower affinity. In contrast to K+, the Li+ inhibition curve is hyperbolic, suggesting interaction at an uncoupled site. Also differing from the complete inhibition by high K+, a residual ouabain-binding rate persists at high Li+. The interactions of Li+ and K+ are synergistic: the apparent K+ affinity increases 3 to 4-fold in presence of Li+. These results are consistent with the conclusion that Li+ interacts with only one of the two K+ sites and may be of interest in interpreting lithium pharmacology.     

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8-12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1, 3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50 = 0.7-0.9 mM), N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.     

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.     

Estimates of Na+-K+ pumping in intact canine iliac arteries

Estimates of Na+ pumping capacity were made using Na+-loaded canine iliac arteries. Ouabain-sensitive uptake of 204Tl or 86Rb was used to measure near-maximal pump rates and [3H]ouabain binding to measure the number of pump sites. Compared with Rb+, Tl+ had the higher affinity for the pump and showed better signal-to-noise characteristics. Maximal uptakes were 0.545 mumol . g-1 . min-1 for Rb+ and 0.40 mumol . g-1 . min-1 for Tl+. Specific ouabain binding (Kd: 28.62 +/- 0.58 nM) was inhibited by external K+, Tl+, and Rb+ and a maximal binding of 51.6 pmol/g wet weight translated into 3.2 X 10(13) sites per gram wet weight. Using these values, the maximal values of K+ transported per pump site per minute lie between 7752 and 10562. If each activation of the pump moves 2K+, the turnover rates could lie between 3876 and 5281 per minute.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

Effect of amiodarone on Na+-, K+-ATPase and Mg2+-ATPase activities in rat brain synaptosomes

Amiodarone hydrochloride is a diiodinated antiarrhythmic agent widely used in the treatment of cardiac disorders. With the increasing use of amiodarone, several untoward effects have been recognized and neuropathy following amiodarone therapy has recently been reported. The present studies were carried out to study the effect of amiodarone on rat brain synaptosomal ATPases in an effort to understand its mechanism of action. Na+, K+-ATPase and oligomycin sensitive Mg2+ ATPase activities were inhibited by amiodarone in a concentration dependent manner with IC50 values of 50 microM and 10 microM respectively. [3H]ouabain binding was also decreased in a concentration dependent manner with an IC50 value of 12 microM, and 50 microM amiodarone totally inhibited [3H]ouabain binding. Kinetics of [3H]ouabain binding studies revealed that amiodarone inhibition of [3H]ouabain binding is competitive. K+-activated p-nitrophenyl phosphatase activity showed a maximum inhibition of 32 per cent at 200 microM amiodarone. Synaptosomal ATPase activities did not show any change in rats treated with amiodarone (20 mg kg-1 day-1) for 6 weeks, when compared to controls. The treatment period may be short, since the reported neurological abnormalities in patients were observed during 3-5 years of treatment. The present results suggest that amiodarone induced neuropathy may be due to its interference with sodium dependent phosphorylation of Na+, K+-ATPase reaction, thereby affecting active ion transport phenomenon and oxidative phosphorylation resulting in low turnover of ATP in the nervous system.     

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.