Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein

The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity.     

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.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group

A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent dephosphorylation of the enzyme without inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation. Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of enzyme labeled with the fluorescent sulfhydryl reagent, 2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+ inhibition of activity, the affinity for Mg2+ as an inducing agent for this effect was significantly reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced in magnitude by ouabain and prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+ (and cations that substitute for K+ in supporting activity) induced a 3 to 4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and ATP, although the K+ and Mg2+ effects appeared to be different on the basis of their excitation spectra. The K+ effect was inhibited by ouabain and occurred with a rate greater than the rate of turnover of the enzyme, permitting its involvement in the catalytic cycle.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover

The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.     

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

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.     

Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.     

The inhibitory monoclonal antibody M7-PB-E9 stabilizes E2 conformational states of Na+,K(+)-ATPase.

Monoclonal antibody M7-PB-E9 binds the sheep kidney Na+,K(+)-ATPase alpha-subunit with high affinity (Kd = 3 nM) and inhibits enzyme turnover in competition with ATP, and, like ATP, in the presence of Mg2+, it stimulates the rate of ouabain binding [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. In this study, covalent attachment of fluorescein 5'-isothiocyanate (FITC) at (or near) the enzyme's ATP binding site did not alter the antibody's affinity for alpha nor did bound antibody alter the anisotropy of (r = 0.36) or the solvent accessibility of iodide to bound FITC. Further, in its E1Na+ conformation (4 mM NaCl), the enzyme's affinity for the ATP congener eosin was unaltered by the bound antibody (Kd = 9 nM). In contrast, partial E2 conformations induced by KCl lowered eosin affinities (0.2 mM KCl, Kd = 28 nM; 0.4 mM, Kd = 86 nM), and M7-PB-E9 reduced these affinities further (Kd = 66 and 130 nM, respectively). By monitoring the fluorescence changes of the FITC-labeled enzyme, the antibody was found to assist several ligand-induced conformational transitions from E1 (E1Na+ or E1Tris) to E2 (E2K+, E2-P(i)Mg2+, or E2Mg2+.ouabain) states, and inhibit the E2K(+)-->E1Na+ transition. Antibody binding alone, however, did not appear to significantly alter enzyme conformation. The antibody therefore is not directed against the ATP site but binds to a region of alpha distinct from any ligand binding site and which plays an important role in the E1<-->E2 transitions.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.     

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.