Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis

An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay

This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains.     

Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions.

The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.     

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.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models

To delineate better the reaction sequence of the (Na+ + K+)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na+ + K+)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after Pi release. However, contrary to requirements of usual formulations, Pi is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K+, and with exclusive binding of substrate vs. either Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-K+ enzyme form occurring in the (Na+ + K+)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E1/E2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E2 conformation as the active site for the phosphatase reaction.     

Ligand binding sites of the ouabain-complexed (Na+ + K+)-ATPase

To clarify the mechanism of inhibition of (Na+ + K+)-ATPase by cardiac glycosides, we tried to see if ouabain binding alters the properties of the binding sites for Na+, K+, and ATP. Ouabain was bound in the presence of either Na+ + MgATP or MgPi. Ligand-induced changes in the rate of release of ouabain from the two resulting complexes were used as signals to determine the affinities, the numbers, and the interactions of the ligand binding sites. Because the two complexes showed differences in the properties of their ligand binding sites, and since neither complex could be converted to the other, it is concluded that either the enzyme has two dissimilar but mutually exclusive ouabain sites or that it can be frozen in two distinct conformations by ouabain. The following ligand sites were identified on the two complexes: 1) two coexisting ATP sites (K0.5 values, 0.1 and 2 mM) representing altered states of the catalytic and the regulatory sites of the native enzyme; 2) mutually exclusive Na+ and K+ sites whose affinities (K0.5 values, 1.3 mM Na+ and 0.1 mM K+) suggested their identities with the high affinity uptake sites of the native enzyme; and 3) coexisting low affinity Na+ and K+ sites (K0.5 values, 0.2-0.6 M) representing either the discharge sites, or the regulatory sites, or the access channels of the native enzyme. The data suggest that the inability of the ouabain-complexed enzyme to participate in the normal reaction cycle is not because of its lack of ligand binding sites but most likely due to ouabain-induced disruptions of interprotomer site-site interactions.     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na+-Na+ exchange of reconstituted shark (Na+ + K+)-ATPase is electrogenic with a transport stoichiometry as for the Na+-K+ exchange, suggesting that translocation of extracellular Na+ is taking place via the same route as extracellular K+. The preparation thus offers an opportunity to compare the sided action of Na+ and K+ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K+. With Na+ but no K+ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K+ in concentrations of 50 microM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K+ also gives biphasic ATP-activation curves, however, only when the K+ concentration is 50 mM or higher (Na+ + K+ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E2(Na2+) and E2(K2+), respectively, and in which the dephosphorylation of E2-P is rate limiting in the presence of Na+ (but no K+) extracellular, whereas in the presence of extracellular K+ it is the deocclusion of E2(K2+) which is rate limiting.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

Kinetic properties of C12E8-solubilized (Na+ + K+)-ATPase

The properties of the rectal gland (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether ( C12E8 ) have been investigated. The kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that Km for ATP for the (Na+ + K+)-ATPase is about 30 microM for the solubilized enzyme and about 100 microM for the membrane-bound enzyme. The Na+-form (E1) and the K+-form (E2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and eosin fluorescence responses to Na+ and K+. The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95-100% pure supernatant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C12E8 /protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C12E8 is required both for solubilization and inactivation. It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C12E8 , and its advantages in the assay of detergent-solubilized (Na+ + K+)-ATPase are discussed. The results show that (Na+ + K+)-ATPase can be solubilized in C12E8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C12E8 -solubilized (Na+ + K+)-ATPase is therefore a good model system for a solubilized membrane protein.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes

Purified (Na+ + K+)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079-3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a Ki of approx. 30 microM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 microM was determined. The addition of the protonophore 1799 and the Me+/H+ exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K0.5 of 700 microM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity (K0.5 less than or equal to 1 mM) and a low-affinity (K0.5 greater than or equal to 0.2 M). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephosphorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same (K0.5 = 2 microM) with regard to the peak currents and the stationary currents. However, for the (Na+ + K+)-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.     

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux.

In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.