ATP binding to solubilized (Na+ + K+)-ATPase. The abolition of subunit-subunit interaction and the maximum weight of the nucleotide-binding unit

Membrane-bound (Na+ + K+)-ATPase from pig kidney outer medulla shows apparent heterogeneity in its ATP-binding site population when assays are carried out in the presence of K+. This finding has been interpreted as being due to interaction between (at least) two subunits, each containing an ATP-binding site. Treating the membrane-bound enzyme with the detergent, C12E8, has been shown to solubilize enzymatically active alpha beta-protomers. We show that in the dissolved enzyme all ATP-binding sites in the population are identical both in the absence and in the presence of K+, which would be consistent with an abolition of identical both in the absence and in the presence of K+, which would be consistent with an abolition of subunit-subunit interaction. This supports previous suggestions that enzyme solubilized by C12E8 is monomeric and that the membrane-bound enzyme is not. Differential extraction of enzyme-containing membranes with C12E8 yielded preparations with an ATP-binding capacity of up to 5.8 nmol per mg protein, measured by the method of Lowry et al. (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275), with bovine serum albumin as standard. Evidence is presented that makes it likely that preparations with an ATP-binding capacity of 7.5 nmol per mg protein (as determined by the above-mentioned assay) will be obtainable. This corresponds to an alpha beta-protomer molecular weight of 133 000 which approximates closely to the minimum value found in the literature for an alpha beta-protomer (i.e., 126 000).     

Radiation inactivation of (Na+ + K+)-ATPase a small target size for the K+-occluding mechanism

Radiation inactivation of partially purified (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb⁺ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for $\text{p-}\text{nitrophenyl phosphatase activity}$, which is itself smaller than the reported target size for (Na⁺ + K⁺)-ATPase activity.     

(Na+ + K+)-ATPase: On the number of the ATP sites of the functional unit

Questions concerning the number of the ATP sites of the functional unit of (Na⁺ + K⁺)-ATPase (i.e., the sodium pump) have been at the center of the controversies on the mechanisms of the catalytic and transport functions of the enzyme. When the available data pertaining to the number of these sites are examined without any assumptions regarding the reaction mechanism, it is evident that although some relevant observations may be explained either by a single site or by multiple ATP sites, the remaining data dictate the existence of multiple sites on the functional unit. Also, while from much of the data it is clear that the multiple sites of the unit enzyme represent the interacting catalytic sites of an oligomer, it is not possible to rule out the existence of a distinct regulatory site for ATP in addition to the interacting catalytic sites. Regardless of the ultimate fate of the regulatory site, any realistic approach to the resolution of the kinetic mechanism of the sodium pump should include the consideration of the established site-site interactions of the oligomer.     

Potassium binding to the (Na+ + K+)-ATPase

The number of K+ bound to the (Na+ + K+)-ATPase has been measured under equilibrium conditions by a differential-titration technique (Hastings, D.F. (1977) Anal. Biochem. 83, 416-432). 5.1 K+ were bound per 32P-labelling site. The K'D for K+ was dependent on the concentration of choline, which was included to give ionic strength. K'D was 59 +/- 2.5 microM with 97 mM choline, 26 +/-1.9 microM with 30 mM choline. The K+ : choline selectivity was 2564 : 1 and the calculated K'D for K+ with zero choline was 11 microM and for choline with zero K+ was 28 mM. 20 microM ATP in the presence of 97 mM choline incresed the K'D for potassium 3-fold to 177 +/- 14 microM. The K'D for K+ with 3 mM Na+ in the presence of 27 mM choline was 81 +/- 10 microM and with 30 mM Na+ without choline 700 +/- 250 microM. The calculated K'D for Na+ at zero K+ and zero choline was 0.6 +/- 0.2 mM. The K+ : Na+ selectivity was 54 : 1.     

Dog kidney (Na+, K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+, K+)-ATPase

(Na⁺, K⁺)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na⁺,K⁺)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K⁺ and Na⁺ at sites where K⁺ promotes and Na⁺ opposes vanadate binding. As a result of Na⁺-K⁺ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na⁺ concentrations in red cell than in kidney (Na⁺,K⁺)-ATPase. It is possible that vanadate could selectively regulate Na⁺ transport in the kidney.     

Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

ATP-binding is stabilized by a stacking interaction within the binding site of Na+/K+ -ATPase

Site-directed mutagenesis was applied to modify phenylalanines (Phe(475)Trp, Phe(548)Tyr, and both) to generate mutants on the basis of molecular modeling of the ATP-binding domain of Na(+)/K(+)-ATPase, in order to characterize the forces that stabilize ATP in its binding pocket. Each of the mutants was examined by Raman difference spectroscopy, i.e., as a difference between the spectrum of the domain with and without bound ATP. It was shown that Phe(475) plays a key role in stabilizing ATP-binding by a stacking interaction. Phe(548) co-stabilizes ATP on the opposite site of the binding pocket and its type of interaction with ATP-binding differs from that of Phe(475).     

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.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase.

K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.     

Coexistence of two ATP sites on the ouabain-complexed (Na+ + K+)-ATPase

When the effects of varying concentrations of ATP on the dissociation rate of the ouabain-enzyme complex were studied, the dissociation rate constant increased with increasing ATP concentrations up to 1 mM, and then decreased with further rise in ATP; indicating that ATP binds to two distinct sites on the complex. ADP and AMP-PNP had similar biphasic effects. GTP, CTP, UTP, and AMP-PCP reduced the dissociation rate. AMP and Pi had no effects. Increase in dissociation rate caused by 0.5 mM ATP was not abolished by saturating CTP, indicating the binding of CTP to only one of the two ATP sites. The data suggest the existence of separate catalytic and regulatory sites, with different affinities and nucleotide specificities.     

Stability of [3H]ouabain binding to the (Na+ + K+)-ATPase solubilized with C12E8

The (Na+ + K+)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [3H]ouabain, solubilized with C12E8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C12E8, Mg2+, HPO4(2-) and glycerol at 20-23 degrees C. The peaks of [3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000-490 000. In addition, these bindings of [3H]ouabain to the (Na+ + K+)-ATPase were observed to be stable at 20-23 degrees C for at least 18 h after the solubilization.     

Pb2+ and imidazole-activated phosphorylation by ATP of (Na+ + K+)-ATPase

In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na++ K+)-activated ATPase

(1) The Mg²⁺-induced low-affinity nucleotide binding by (Na⁺ + K⁺)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the K_d value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na⁺ + K⁺)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (K_m ATP) and for the true affinity in the binding of AdoPP[NH]P (K_d and K_i) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the K_m value, which thus is smaller than the K_d value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the K_d value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the K_d value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the K_d value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

The K+-induced apparent heterogeneity of high-affinity nucleotide-binding sites in (Na+ + K+)-ATPase can only be due to the oligomeric structure of the enzyme

K⁺ induces an apparent heterogeneity among an otherwise homogeneous population of nucleotide-binding sites in (Na⁺ + K⁺)-ATPase preparations from pig kidney. With the help of ouabain we show that this heterogeneity cannot be due to a mixture of different and independent sites and conclude that each enzyme molecule must contain two nucleotide site-containing units that show interaction. Na⁺ induces an apparent heterogeneity among an otherwise homogeneous population of ouabain-binding sites. The argument is, therefore, extended to include one ouabain site on each of the structural units that bind nucleotide. All these structural units are shown to hydrolyse substrate at identical rates. Using the presently available molecular weight data, it is concluded that the enzyme is composed of two subunits each possessing one nucleotide-binding site, one ouabain-binding site, one α-peptide and the capacity for hydrolysing ATP and $\text{p-}\text{nitrophenyl}$ phosphate.     

Na+, K+-ATPase: evidence for the binding of ATP to the phosphoenzyme

Highly purified Na⁺, K⁺-ATPase of the dog kidney was reacted with Mg²⁺+³²Pi or Mg²⁺+³²Pi + ouabain. ³²P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K_0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na⁺. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested.