Electrical potential accelerates the E1P(Na)----E2P conformational transition of (Na,K)-ATPase in reconstituted vesicles

We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out-oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle.     

Uncoupling the red cell sodium pump by proteolysis

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.     

Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase

The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).     

The regulation of Na, K-ATPase activity by the substrate

The mechanism of functioning of Na, K-ATPase system is considered, the peculiarities of hydrolysis in different substrates are described. The experimental results testify to the role of substrate structure in E2----E1-transition, Na+ transport, K(+)-dependent phosphatase activity and quaternary structure of enzyme. The regulatory role of molecular organization of Na, K-ATPase in ion transport is discussed.     

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.     

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.     

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.     

Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate

The rate of 86Rb or 42K release from an occluded form of the phosphorylated Na+ pump has been studied using a rapid filtration apparatus described previously. The rate constant of release is 5-15 s-1, and 42K and 86Rb dissociate at approximately the same rate. Mg2+ is required for deocclusion in the presence of Pi at a site which has the same affinity as the site involved in stabilization of E2(K) with ATP; we propose that Na,K-ATPase has only one site for Mg2+ (apart from Mg2+ complexed with ATP), that the affinity of this site for Mg2+ is increased by Pi binding and decreased by ATP binding, and that Mg2+ is bound and released in the normal transport cycle. In the presence of K+, Cs+, Rb+, or Tl+, the release of two distinct 86Rb ions can be observed, the slow release from one site ("s" site) being blocked by occupancy of the site vacated by the other ("f", fast site). By a sequence of incubations, labeled 86Rb can be placed at either site, and the rate of dissociation monitored individually; in the absence of K+, dissociation from the s site proceeds after a lag in which the f site is vacated. The results are consistent with a "flickering-gate" model of deocclusion to the extracellular pump face, in which the site is exposed to the medium only long enough for a single ion to be released. When deocclusion to the intracellular face is promoted with ATP, ions are released from both sites at the same rate, presumably because the E2----E1 conformational change is rate-limiting. Unlabeled ions co-occluded with 86Rb increase the ATP-stimulated rate of release in the order Rb+ less than Tl+ less than Cs+ less than K+; since the same rank order is observed when dissociation from the s site is monitored in the presence of these ions and MgPi we propose that the latter process proceeds toward the intracellular pump face. 86Rb release from the vanadate-inhibited enzyme has the characteristics of Pi-stimulated release but is approximately 25-fold slower. ATP binds to both the phosphorylated and vanadate-inhibited forms of Na,K-ATPase and increases the rate of deocclusion, apparently to both the intracellular and extracellular faces of the pump.     

Phosphoenzyme decomposition in dog cardiac sarcoplasmic reticulum Ca2+-ATPase

A five-syringe quench-flow apparatus was used in the transient-state kinetic study of intermediary phosphoenzyme (EP) decomposition in a Triton X-100 purified dog cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase at 20 degrees C. Phosphorylation of the enzyme by ATP in the presence of 100 mM K+ for 116 ms gave 32% ADP-sensitive E1P, 52% ADP- and K+-reactive E2P, and 16% unreactive residual EPr. The EP underwent a monomeric, sequential E1P 17 s-1----E2P 10.5 s-1----E2 + Pi transformation and decomposition in the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid quenched Ca2+-devoid medium. The calculated rate constant for the total EP (i.e., E1P + E2P) dephosphorylation was 7.8 s-1. The E1P had an affinity for ADP with an apparent Kd congruent to 100 microM. When the EP was formed in the absence of K+ for 116 ms, no appreciable amount of the ADP-sensitive E1P was detected. The EP comprised about 80% ADP- and K+-reactive E2P and 20% residual EPr, suggesting a rapid E1P----E2P transformation. Both the E2P's formed in the presence and absence of K+ decomposed with a rate constant of about 19.5 s-1 in the presence of 80 mM K+ and 2 mM ADP, showing an ADP enhancement of the E2P decomposition. The results demonstrate mechanistic differences in monomeric EP transformation and decomposition between the Triton X-100 purified cardiac SR Ca2+-ATPase and deoxycholate-purified skeletal enzyme [Wang, T. (1986) J. Biol. Chem. 261, 6307-6319].     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

Inactivation of Na,K-ATPase following Co(NH3)4ATP binding at a low affinity site in the protomeric enzyme unit

The Na(+)-dependent or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP, but submillimolar concentrations are needed to accelerate the K(+)-dependent or E2 half of the cycle. Here we use Co(NH(3))(4)ATP as a tool to study ATP sites in Na,K-ATPase. The analogue inactivates the K(+) phosphatase activity (an E2 partial reaction) and the Na,K-ATPase activity in parallel, whereas ATP-[(3)H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH(3))(4)ATP binding (K(D) approximately 0.4-0.6 mm), we can also measure high affinity equilibrium binding of Co(NH(3))(4)[(3)H]ATP (K(D) = 0.1 micro m) to the native enzyme. Crucially, we find that covalent enzyme modification with fluorescein isothiocyanate (which blocks E1 reactions) causes little or no effect on the affinity of the binding step preceding Co(NH(3))(4)ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH(3))(4)ATP bind within different enzyme pockets. The Co(NH(3))(4)ATP enzyme was solubilized with C(12)E(8) to a homogeneous population of alphabeta protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH(3))(4)ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (alphabeta)(2) membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized alphabeta protomer could be directly inactivated by Co(NH(3))(4)ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH(3))(4)ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.     

The role of the substrate structure in the function of Na,K-ATPase

The mechanism of Na,K-ATPase function is reviewed. The peculiarities of hydrolysis of various substrates are described. The experimental results testify to the effect of substrate structure on the E2----E1 transition, rate of Na+ transport, K-dependent phosphatase activation and the quaternary structure of Na,K-ATPase. A conclusion is drawn that the proton-acceptor properties of the substrate play a role in the regulation of ion transport by Na,K-ATPase.     

Characterization of lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase.

Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Fluorescent labeling of (Na+ + K+)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na+ + K+)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K+-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na+, K+, Mg2+, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na+, and Mg2+, and decreased by K+. These fluorescence changes likely reflect ligand-induced stabilization of the E1 or E2 states of the enzyme.     

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.     

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.     

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.     

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.