Inactivation of Na,K-ATPase Following Co(NH3)4ATP Binding at a Low Affinity Site in the Protomeric Enzyme Unit*

Douglas G. WardDagger and José D. Cavieres§

From the Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester LE1 9HN, United Kingdom

Received for publication, October 30, 2002, and in revised form, February 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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(NH3)4ATP 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-[3H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH3)4ATP binding (KD approx  0.4-0.6 mM), we can also measure high affinity equilibrium binding of Co(NH3)4[3H]ATP (KD = 0.1 µM) 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(NH3)4ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH3)4ATP bind within different enzyme pockets. The Co(NH3)4ATP enzyme was solubilized with C12E8 to a homogeneous population of alpha beta protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH3)4ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (alpha beta )2 membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized alpha beta protomer could be directly inactivated by Co(NH3)4ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH3)4ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.

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ABSTRACT
INTRODUCTION
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The sodium pump or Na,K-ATPase1 is the enzyme that mediates active transport of Na+ and K+ across the plasma membrane of animal cells (1-3). It consists of an alpha  or catalytic subunit (Mr 112,000) and a beta  subunit, a glycoprotein (protein Mr 35,000) found in a 1:1 ratio (2). The alpha beta heterodimer, or alpha beta protomer, appears as the minimal catalytically active unit after solubilization with C12E8 (4-6) and probably also represents the minimal enzymic unit in the membrane (7, 8). There is general agreement that the reaction cycle of Na,K-ATPase and other P-type ATPases proceeds through the formation and breakdown of phosphoenzyme intermediates (9, 10), as the enzyme cycles between an E1 or Na+ form, and an E2 or K+ form (11).

Both transport and ATP hydrolysis by Na,K-ATPase have complex responses to ATP (12, 13). This behavior is also observed with the alpha beta protomer (6) and seems to result from two types of ATP interaction with the enzyme. On the one hand, the E1 (Na+-dependent) reactions are saturated at a few micromolar ATP and are associated with phosphorylation of the alpha -chain at Asp369 (2, 14). In the case of Na,K-ATPase, the next documented ATP requirement only arises after hydrolysis of the phosphoenzyme; it consists of an acceleration of K+ release from the "occluded K+ form" to the intracellular medium, a rate-limiting step (9, 15, 16), and results in a large stimulation of pump activity. Here ATP acts with a low affinity, with K0.5 in the region of 0.2-0.5 mM (15), and can be replaced by ADP (17) and non-phosphorylating analogues of ATP (18). The requirements and affinities of E1 and E2 reactions largely explain the apparent negative co-operativity in the ATP activation of Na,K-ATPase activity and Na+-K+ exchange. Nucleotides also show low affinity inhibitory effects toward some E2 reactions of Na,K-ATPase. Among these are the hydrolysis of the phosphoenzyme formed from 32Pi and the K+ phosphatase activity, i.e. the ability to hydrolyze synthetic substrates, like p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate, in the presence of K+ (19, 20).

Several laboratories have suggested the co-existence of separate high affinity and low affinity ATP sites in P-type ATPases (for review, see Ref. 21). One of the paradigms is that of FITC, which binds covalently to alpha Lys501 in Na,K-ATPase (22) and Lys515 in SERCA (23), in what now clearly appears as a high affinity adenine-binding site; in the 2.6-Å SERCA structure (24) Lys515 is located deep inside a nucleotide binding pocket in the N domain. FITC blocks E1 functions like high affinity ATP binding and [gamma -32P]ATP phosphorylation, as well as the overall (Na,K-ATPase) activity (25-27). On the other hand, E2 functions like 32Pi phosphorylation and dephosphorylation, and the K+ phosphatase activity, are little affected by the FITC modification; the latter is true whether the substrate is acetyl phosphate, pNPP, or 3-OMPF (25-29, 73). In the FITC-enzyme, both the K+ phosphatase activity and dephosphorylation of the phosphoenzyme formed with 32Pi are inhibited by ATP and TNP-ADP, with low affinities (28, 29). We have reported that FITC can access all alpha -chains of membrane-bound Na,K-ATPase, that pNPP and 3-OMFP can be split by the solubilized, FITC-modified alpha beta protomer, and that TNP-ADP can inhibit and TNP-8N3-ADP photoinactivate the K+ phosphatase activity of the FITC enzyme (29, 30). This suggested that TNP-ADP should bind at a low affinity nucleotide site on the FITC-modified alpha -chain.

The experiments in the present paper make use of Co(NH3)4ATP, a substitution-inert ATP analogue (31-34) with actions complementary to those of FITC. Following equilibrium binding at a low affinity site, Co(NH3)4ATP becomes trapped, albeit non-covalently, and inactivates Na,K-ATPase by blocking E2 reactions but not E1 steps (31, 32). Interestingly, after incorporating a maximal quantity of Co(NH3)4[gamma -32P]ATP in the membrane enzyme, it was still possible to bind an equivalent amount of Cr[gamma -32P]ATP but not Co(NH3)4[32P]PO4 (34). Our results show that Co(NH3)4ATP can access all alpha beta protomeric units, either in detergent solution or in the membrane, and that the survival of E1 functions can be best explained on the basis of separate high affinity and low affinity ATP sites on the alpha -chain.

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Enzyme Purification-- Na,K-ATPase was purified in membrane-bound form from pig kidney outer medulla, by using the zonal-rotor procedure of Jørgensen (35), and stored at 0 °C in 25 mM imidazole, 1 mM EDTA (pH 7.4). The specific activity ranged from 20 to 35 µmol min-1 mg protein-1.

Co(NH3)4ATP Synthesis-- This was carried out according to Cornelius et al. (36). The purity of the product was assessed by high performance liquid chromatography on a Partisil 10 SAX column (see under "ATP-ADP Exchange"), where Co(NH3)4ATP eluted at 4:24 min (Fig. 1A). The elution time is similar to that of AMP (Fig. 1B), as expected for the bidentate complex (37). The concentration of the product was assessed from the UV and visible spectrum and the molar extinction coefficients at 257, 366, or 518 nm (36). Co(NH3)4[3H]ATP was synthesized from [2,8-3H]ATP in the same manner, to specific activities between 500 and 3000 MBq mmol-1. The purified products were stored in aliquots at -80 °C.


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Fig. 1.   HPLC of adenine nucleotides. The samples were run on a Partisil 10 SAX column (0.46 × 25 cm) with A = 50 mM ammonium formate (pH 4.6) and B = 1.4 M ammonium phosphate (pH 3.7). The straight line segments show the gradient in % B, recorded simultaneously with offset compensation. A, Co(NH3)4ATP. One ml was injected; the 260 nm trace was obtained with a 20 µM sample and the 518 nm trace with a 1 mM sample, and both runs were superimposed on the same chart. B, AMP, ADP, and ATP. Twenty nmol of each (1 ml) in 5 mM Tris/Cl (pH 7.5), monitored at 260 nm. Horizontal bars show collection windows for the fractions containing AMP + Pi (M), ADP (D), and ATP (T).

Inactivation of Na,K-ATPase with Co(NH3)4ATP-- Aliquots of purified Na,K-ATPase (at about 1 mg of protein ml-1) were incubated for up to 3 h at 37 °C in 20 mM imidazole (pH 6.8), with up to 3 mM Co(NH3)4ATP or without (controls). In most experiments, the enzyme samples were cooled down and centrifuged for 15 min at 425,000 × g and 5 °C in a Beckman TL100 benchtop ultracentrifuge. The membranes were resuspended in chilled 20 mM imidazole (pH 6.8) with the help of a Dounce homogenizer, washed once by ultracentrifugation, and resuspended in the same medium. Protein and enzymic activities were then determined in inactivated and control suspensions. In the experiment shown in Fig. 5, the incubation at increasing Co(NH3)4ATP concentrations was made in 30 µl, and 4-µl aliquots were diluted into 0.8 ml of K+ phosphatase medium (containing 3-OMFP) in a spectrophotometer cuvette, and the K+ phosphatase reaction was followed as described below. To analyze the Co(NH3)4ATP concentration effect, the inactivation rate constant kinact was first calculated as the negative slope of plots of the natural logarithm of the fractional activity left against time, and the results were replotted against the Co(NH3)4ATP concentration (38).

Inactivation with FITC-- This was done essentially as described (29), at protein concentrations between 1 and 2.5 mg/ml and in a medium containing 150 mM NaCl, 50 mM Tris/Cl- (pH 9.2), 5 mM EDTA, and 0.5% dimethyl sulfoxide. Parallel enzyme samples were incubated with or without 20 µM FITC for up to 4 h at 20 °C, spun down, washed, and resuspended with 20 mM Tris/Cl- (pH 7.5), 2 mM EDTA, 2 mM dithiothreitol, or with 40 mM imidazole (pH 6.8), 2 mM EDTA.

Equilibrium Binding of Co(NH3)4[3H]ATP or [gamma -33P]ATP-- Equal volumes of a Na,K-ATPase suspension and radioligand solution were mixed and incubated with stirring for 20 min at 20 °C so that the final concentrations were as follows (mM): NaCl 150, TES 10 (pH 7.2), dithiothreitol 1, EDTA 1. At the end of this period, a volume was extracted by means of a syringe (27) fitted with a µStar® cellulose-nitrate filter (Costar, 0.2-µm pore diameter). One ml of the filtrate ("free nucleotide") was counted in a Beckman LS-6000-TA scintillation spectrometer side by side with 1 ml of the suspension ("total nucleotide"), with quench, chemiluminescence, and decay corrections and against calibrated Co(NH3)4[3H]ATP or [gamma -33P]ATP standards. Bound nucleotide was calculated from total and free nucleotide measurements and referred to the protein mass of the sample.

Na,K-ATPase Solubilization to alpha beta Protomers-- The purified enzyme was solubilized as already described (4-6). Briefly, samples of control and Co(NH3)4ATP-treated Na,K-ATPase were washed by ultracentrifugation and resuspended with a medium containing 300 mM NaCl and 40 mM TES (adjusted to pH 7.4-7.5 with NaOH). The protein concentration was determined and then adjusted to 680 µg ml-1; an equal volume of C12E8 solution was added to achieve a final detergent concentration of 1 mg ml-1. After 30 min at room temperature, the samples were ultracentrifuged at 435,000 × g for 15 min and the pellets discarded. Enzymic assays were performed as described below, except that C12E8 was added at the critical micelle concentration (50 µg ml-1) to all reaction media.

Analytical Ultracentrifugation of the Solubilized Enzyme-- Sedimentation velocity runs were carried out at 40,000 rpm and 20 °C on a Beckman Optima XL-A analytical ultracentrifuge (6) (National Centre for Macromolecular Hydrodynamics, Leicester and Nottingham Universities, UK). The enzyme was solubilized with C12E8 as described above, and the high speed supernatants of control and Co(NH3)4ATP-inactivated enzymes were analyzed in the same run. The double-sectored cells were scanned radially at 280 nm at 20- or 30-min intervals; all samples showed a single transition zone. The sedimentation coefficient s20,b (at 20 °C in buffer) and its intrinsic error were calculated from linear regression on the natural logarithm of the radial distance of the midpoint of the depletion region versus time and corrected to s20,w (39). The protein molecular mass of the solubilized enzyme was calculated according to Tanford et al. (40), using a Stokes radius of 72 Å and a partial molar volume = 0.646 cm3 g-1 (4, 5).

Na,K-ATPase Determinations-- ATP hydrolysis was measured from the release of 32Pi from [gamma -32P]ATP, as described previously (6). All enzymic assays were routinely conducted at 20 °C and as time courses with at least 6 time points over 10 min. Na,K-ATPase reactions were carried out in 50-µl samples of a medium containing (mM) the following: [gamma -32P]ATP 1, MgCl2 1.5, NaCl 130, KCl 20, imidazole 20 (pH 7.2), plus membrane-bound or soluble enzyme at 20-30 µg ml-1. The reactions were started by mixing components pre-equilibrated at 20 °C and stopped by immersing samples in a dry ice/ethanol bath. After defrosting at 0 °C, 32Pi was extracted as phosphomolybdate into isobutyl alcohol/ethyl acetate (1:1), side by side with acid-hydrolyzed standards, and aliquots were counted in a scintillation spectrometer.

K+ Phosphatase Activity-- This was evaluated in either of two ways. To measure the release of p-nitrophenol from pNPP (29) at 20 or 37 °C, the medium routinely contained (mM) the following: pNPP 10, MgCl2 6, KCl 150, imidazole 20 (pH 7.2), plus enzyme at 20-30 µg ml-1 (the pNPP concentration was varied in the experiments shown in Fig. 4). Reaction samples (50 µl) were stopped by dilution into 300 µl of 0.1 M NaOH, and the absorbances were read at 410 nm in a Beckman DU65 spectrophotometer against a p-nitrophenol calibration curve. In the experiment shown in Fig. 5, instead, the medium above contained 0.5 mM 3-OMFP instead of pNPP, and the release of 3-OMF was followed for up to 300 s at 475 nm and 37 °C. This was done in thermostatted, stirred 1-ml cuvettes in a U-3310 Hitachi spectrophotometer fitted with a 6-cell changer (cycling time, 30 s; sampling time, 3 s). All measurements were corrected for spontaneous 3-OMFP hydrolysis. As with pNPP, all phosphatase activities were obtained from linear regressions on at least 6 time points of product release against time.

ATP-ADP Exchange-- The rate of reversible initial phosphorylation of Na,K-ATPase was measured at 20 °C from the backward half-reaction, as incorporation of the [2,8-3H]ADP label into ATP, and was corrected for [gamma -32P]ATP hydrolysis (41). The reactions were generally carried out in a medium containing (mM) the following: [2,8-3H]ADP 1.25, [gamma -32P]ATP 1, MgCl2 1.6 (400 µM calculated Mg2+), NaCl 10, imidazole 20 (pH 7.2). Samples (10-20 µl) were stopped by mixing with 5 µl of 120 mM EDTA at room temperature and immersing in a dry ice/ethanol bath. Defrosted samples and "initials" were mixed with chilled ATP/ADP/AMP/Pi carriers, and the nucleotides and Pi were separated on a Partisil 10 SAX column (25 × 0.46 cm), using an Amersham Biosciences HPLC system (Fig. 1B). The separation was optimized for base-line resolution and speed, with an elution rate at 1.5 ml min-1, and the following gradient (min:s/% B): 0:00/0%, 0:01/15%, 5:40/50%, 5:45/100%, 16:00/100%, 16:01/0%, and 22:01/0%; where A is 50 mM ammonium formate (pH 4.6) and B is 1.4 M ammonium phosphate (pH 3.7). The elution times (and collection times in parentheses) in min:s are as follows: AMP/Pi 4:27 (3:55-5:55), ADP 6:26 (5:55-7:55), and ATP 9:25 (8:45-10:45). The eluted fractions (3 ml), plus 12 ml of scintillation fluid (Quicksafe-A, Zinsser Analytic) and 3 ml ethanol (41), were counted for 3H and 32P with quench, decay, and chemiluminescence corrections, together with standards and initial samples.

Protein Determinations-- Protein was determined in 0.5-ml samples, at least in triplicate, using a modified bicinchoninic acid method (6, 42) with bovine serum albumin as standard.

Source of Materials-- ATPNa2 and ADP (free acid) were from Roche Molecular Biochemicals; [2,8-3H]ATP, [2,8-3H]ADP, [gamma -32P]ATP, and [gamma -33P]ATP were purchased from PerkinElmer Life Sciences, and FITC was from Molecular Probes or Sigma. [Co(III)(NH3)4CO3]NO3 was synthesized according to Schlessinger (43). Bicinchoninic acid was from Pierce. HPLC columns were purchased from BASTechnicol (Stockport, Cheshire, UK). pNPP (Tris salt), 3-OMFP, and 3-OMF were from Sigma. All other reagents were of the maximal purity available.

Data Handling-- In Na,K-ATPase and K+ phosphatase assays, the reaction time courses were fitted by least squares linear regression to obtain the enzyme activities and their standard errors. The calculation of the rate of ATP-ADP exchange took the fractional ATP hydrolysis into account; the rate constant for the reverse half-reaction was obtained from the time course data, fitted as a linear transform for exponential approach to isotopic equilibrium (41). Where relative (or percent) specific activities are shown, the errors have been compounded to incorporate the error on the specific activity of the reference sample. Other linear and non-linear curve fitting were done using SigmaPlot 4 (Corte Madera, CA).

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Co(NH3)4ATP-binding Sites-- Two characteristic features of the Co(NH3)4ATP inactivation of Na,K-ATPase are the selective suppression of E2 reactions and the distinctive low affinity of the Co(NH3)4ATP binding step preceding it (31, 32). The experiment in Fig. 2 confirms the first of these attributes. Here we have measured the overall reaction (Na,K-ATPase activity) and two partial reactions, ATP-[3H]ADP exchange (an E1 reaction) and the K+ phosphatase activity (an E2 reaction). ATP-[3H]ADP exchange results from a reversal of the initial enzyme phosphorylation, as the 3H label becomes distributed into the ATP compartment. It is apparent that when the membrane-bound sodium pump is exposed to Co(NH3)4ATP at 37 °C, and then washed by ultracentrifugation, it loses its K+ phosphatase and Na,K-ATPase activities at the same pace. In our hands, the ATP-ADP exchange activity of the Co(NH3)4ATP-treated enzyme returns somewhat variable results, but its survival value is always substantially higher than those of the K+ phosphatase or Na,K-ATPase activities. In the experiment shown in Fig. 2, there was no loss of exchange by the time the other two activities had fallen to a few percent of their controls. Fig. 3A shows how the pseudo-first order rate constant for inactivation of the Na,K-ATPase activity increases with the Co(NH3)4ATP concentration. The hyperbolic dependence confirms that this is a two-step process (31, 38), i.e. that saturable low affinity Co(NH3)4ATP binding at a single enzyme site must occur before Co(NH3)4ATP occlusion and enzyme inactivation. A dissociation constant of 0.62 mM could be fitted to the data, similar to the value reported earlier (31). This is in the concentration range for low affinity ATP effects on the sodium pump, as found when measuring Na,K-ATPase activity (13) or E2 reactions like K+:K+ exchange (18, 44) and K+ (Rb+) occlusion (15).


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Fig. 2.   Selective Co(NH3)4ATP inactivation of E2 function of membrane-bound Na,K-ATPase. Eight enzyme samples were incubated in 1 mM imidazole (pH 6.8) for a total of 3 h at 37 °C, and Co(NH3)4ATP was added to a final concentration of 1 mM, timed to achieve the inactivation periods shown. The samples were cooled down, centrifuged (15 min at 435,000 × g), washed once, and resuspended with 1 mM imidazole (pH 6.8), and their protein concentration and enzymic activities were determined. The plots show the Na,K-ATPase (open circles), K+ phosphatase (solid circles), and ATP-ADP exchange activities (squares), all assayed as time courses at 20 °C, as described under "Experimental Procedures"; vertical bars show standard errors. The control values were (milliunits mg-1 ± S.E.) 1590 ± 40 (Na,K-ATPase), 536 ± 18 (K+ phosphatase), and 4.42 ± 0.06 (ATP-ADP exchange).


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Fig. 3.   High and low affinity Co(NH3)4ATP binding. A, Co(NH3)4ATP concentration dependence of the rate of inactivation of membrane-bound Na,K-ATPase. The enzyme was preincubated at 37 °C in 30 mM imidazole (pH 7.2), 1 mM EDTA, with or without Co(NH3)4ATP at 8 concentrations between 1 µM and 3 mM, and 5 timed samples were drawn within 30 (below 30 µM) or 20 min (between 0.1 and 3 mM). In all 8 × 5 resulting reactions, the Na,K-ATPase activity was determined as linear time courses, and inactivation rate constants kinact were calculated from semilog plots of activity remaining versus time of incubation with Co(NH3)4ATP. The Co(NH3)4ATP concentration dependence of kinact was fitted to the hyperbola shown, with KD = 0.62 ± 0.11 mM and kinact(max) = 2.40 ± 0.18 h-1 (mean ± S.E.). B, concentration dependence of high affinity Co(NH3)4[3H]ATP and [gamma -33P]ATP equilibrium binding to native Na,K-ATPase. In separate experiments, the enzyme (15 µg ml-1) was equilibrated with various concentrations of the radioactive nucleotide at 20 °C, and in a medium containing (mM) NaCl 150, TES (pH 7.2) 10, dithiothreitol 1, EDTA 1. The total and free labeled nucleotide concentrations were determined as described under "Experimental Procedures"; the bound nucleotide on left-hand and right-hand ordinates are referred to the protein concentration. In experiment 1 (solid circles, long-dashed line, left-hand axis), the hyperbola B(ound) = Bmax/(1 + KD/[free]) was fitted to the data, with Bmax = 1.80 ± 0.22 nmol mg-1 and KD = 0.10 ± 0.02 µM; in experiment 2 (open circles, solid line, left-hand axis), the continuous line was fitted with Bmax = 1.75 ± 0.15 nmol mg-1 and KD = 0.11 ± 0.02 µM. For comparison, the lower curve (squares, short-dashed line, right-hand axis) shows equilibrium [gamma -33P]ATP binding to native enzyme, fitted with Bmax = 1.47 ± 0.10 nmol mg-1 and KD = 0.24 ± 0.03 µM (mean ± S.E.).

It has also been reported that Co(NH3)4ATP can act as a competitor in experiments to measure high affinity equilibrium ATP binding (31, 33) but without permanent deleterious effects on the enzyme. We have now made direct measurements of high affinity equilibrium Co(NH3)4ATP binding; the experiments shown in Fig. 3B demonstrate that, with KD = 0.10 µM, Co(NH3)4ATP is as good a ligand as ATP in these conditions (cf. Ref. 45) if not better. Incidentally, given the concentration dependence and temperature in Fig. 3A, and as either Na+ or K+ inhibit the enzyme modification by Co(NH3)4ATP (31), in the equilibrium binding experiments of Fig. 3B (at 20 °C) there must have been little or no enzyme inactivation. To summarize, one may conclude that, with non-cycling Na,K-ATPase, it is possible to observe high affinity Co(NH3)4ATP binding directly and low affinity Co(NH3)4ATP binding at least indirectly.

Complementary Co(NH3)4ATP and FITC Modifications-- The primary Co(NH3)4ATP interaction with the K+ phosphatase reaction is such that the analogue behaves as a competitive inhibitor with respect to pNPP, with Ki = 0.22 mM (33). We have now looked at the consequences of Co(NH3)4ATP inactivation. The experiment shown in Fig. 4A compares the pNPP concentration dependence before and after the enzyme was inactivated with, and washed free from, Co(NH3)4ATP. Whereas K0.5 shows no increase, the maximal K+ phosphatase rate has been reduced considerably, to 12 ± 2%. This is not too different from the residual level of Na,K-ATPase activity and suffices to explain the parallel inactivation time courses seen in Fig. 2. The obvious conclusion is that, rather than reducing the pNPP affinity, the Co(NH3)4ATP modification fully prevents the K+ phosphatase domain from binding or hydrolyzing its substrate. As the irreversible effect is preceded by low affinity Co(NH3)4ATP binding to the enzyme, and as the ATP-ADP exchange activity is unaffected (Fig. 2), a plausible conclusion is that the low affinity Co(NH3)4ATP-binding site and the K+ phosphatase site are different from the site that supports enzyme phosphorylation by ATP, a notion that is reinforced by the results shown in Fig. 5. Fig. 4B shows a contrasting experiment, carried out with FITC-modified Na,K-ATPase. The FITC modification increased K0.5 for pNPP about 2-fold but the maximal K+ phosphatase rate was more or less unaffected, as reported previously (26, 29). The FITC modification does not hinder 32Pi phosphorylation either, but it prevents high affinity ATP binding and phosphorylation by micromolar [gamma -32P]ATP (27, 28). The covalently bound FITC would then be blocking the high affinity ATP site, at least its adenine-binding region (46). Large ATP concentrations during assays with the FITC-modified enzyme fail to increase the low remaining level of Na,K-ATPase activity (28, 29).


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Fig. 4.   Effects of Co(NH3)4ATP and FITC modifications on the K+-phosphatase reaction. A, effect of Co(NH3)4ATP inactivation of native sodium pump on the substrate concentration dependence of pNPP hydrolysis. Enzyme samples were incubated with or without 1 mM Co(NH3)4ATP for 3 h at 37 °C, washed, and resuspended with 20 mM Tris/HCl (pH 7.2). The specific Na,K-ATPase activity of the diluted treated enzyme was 4.9 ± 0.7% of the parallel control. The data show the K+ phosphatase activities obtained from linear time courses of p-nitrophenol release at increasing pNPP concentrations. To facilitate comparison, the data for the control enzyme (open symbols) is plotted against the left-hand axis and that for the Co(NH3)4ATP enzyme (solid symbols) is plotted against the expanded right-hand axis. The hyperbolae were fitted with Vmax (µmol min-1 mg-1) and K0.5 (mM) 0.75 ± 0.03 and 4.3 ± 0.5 (control enzyme, continuous line), and 0.09 ± 0.01 and 3.9 ± 1.1 (Co(NH3)4ATP-inactivated enzyme, dotted line). B, effect of FITC inactivation of native sodium pump. Parallel samples of native enzyme were incubated with or without 20 µM FITC for 4 h at 20 °C, spun down, washed, and resuspended with 200 mM Tris/HCl (pH 7.5), 2 mM EDTA, 2 mM dithiothreitol. The specific Na,K-ATPase activity left was 8.0 ± 0.6% of the parallel control. The specific K+ phosphatase activities shown have been fitted with Vmax (µmol min-1 mg-1) and K0.5 (mM) 0.96 ± 0.02 and 5.4 ± 0.3 (control enzyme, open symbols), and 0.90 ± 0.02 and 11.4 ± 0.4 (FITC-inactivated enzyme, solid symbols), respectively. Standard errors are contained within the symbols.


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Fig. 5.   Co(NH3)4ATP inactivation of the K+ phosphatase activities of native and FITC-modified Na,K-ATPase in a simultaneous experiment. Na,K-ATPase (1.1 mg ml-1) was incubated for 2.5 h at 20 °C, pH 9.2, with or without 20 µM FITC, washed twice by ultracentrifugation, and resuspended with 40 mM imidazole (pH 6.8), 2 mM EDTA. The Na,K-ATPase and K+ phosphatase activities of the FITC-enzyme were 2 and 77%, respectively, of the control enzyme activities. FITC enzyme samples were mixed with an equal volume of water or of 1 of 5 Co(NH3)4ATP dilutions in water. After sampling for zero time K+ phosphatase activities, the tubes (30 µl) were incubated at 37 °C for up to 80 min; six control enzyme mixes were then started 11 min later. Four µl of each of the 12 suspensions were sampled at 0, 20, 50, and 80 min, into 1 ml of K+ phosphatase medium containing 3-OMFP at 37 °C in a spectrophotometer cuvette, and 3-OMF release was followed as described under "Experimental Procedures." Top panel, control enzyme. Samples incubated with Co(NH3)4ATP at a final concentration of (mM) 0.15 (circles), 0.40 (reversed triangles), 0.75 (squares), 1.5 (triangles) and 3.0 (diamonds). The K+ phosphatase activity of each sample has been divided by the activity of the zero Co(NH3)4ATP sample (not shown), and the natural logarithm of the ratio has been plotted against inactivation time. The straight lines have been fitted by linear regression to extract the inactivation rate constant kinact (1/slope). Middle panel, FITC-inactivated enzyme. Co(NH3)4ATP concentrations, data treatment, and symbols as in top panel. Bottom panel, the kinact values obtained in the above panels have been plotted against the Co(NH3)4ATP concentration. Hyperbolae of the form kinact = kinact(max)/(1 + Km/[Co(NH3)4ATP]) were fitted with Km and kinact(max): 0.45 ± 0.11 and 2.11 ± 0.16 h-1 (control enzyme) and 0.41 ± 0.10 mM and 1.71 ± 0.13 h-1 (FITC-enzyme).

The complementarity of Co(NH3)4ATP and FITC modifications had suggested that the enzyme could accommodate both ligands at the same time, in different pockets (47). We treated the enzyme with FITC until its Na,K-ATPase activity had been reduced to 1% that of a parallel control enzyme; its K+ phosphatase activity, measured with pNPP, remained at 75%. When the FITC-modified and control enzymes were incubated side by side with 1 mM Co(NH3)4ATP, the rate constants for inactivation of their K+ phosphatase activities were 0.75 ± 0.02 and 0.86 ± 0.01 h-1, respectively (not shown). As the bulky fluorescein moiety bound to the alpha -chain caused less than 15% decrease in the overall effectiveness of the Co(NH3)4ATP inactivation, it would appear as if FITC did not interfere with the binding of the ATP analogue. However, although unlikely, the result could have arisen from fortuitous compounding of a lower binding affinity with a higher maximal inactivation rate in the FITC enzyme. We compared, therefore, the Co(NH3)4ATP binding affinity and the maximal inactivation rates of native and FITC-modified enzymes. This was first done separately and then as a single experiment where the FITC enzyme and its native control were incubated side by side at various times and Co(NH3)4ATP concentrations (Fig. 5). The design was such that the Co(NH3)4ATP treatment of every enzyme sample was stopped as its spectrophotometric K+ phosphatase assay was started (3-OMFP was used as substrate). The results in Fig. 5 show that there is not much change in K0.5 for Co(NH3)4ATP and only a 20% reduction in the maximal inactivation rate. We should recall that the FITC modification inactivates E1 functions like high affinity ATP binding and phosphorylation by [gamma -32P]ATP as much as the Na,K-ATPase activity (25, 27). In this experiment, therefore, Co(NH3)4ATP is revealing a low affinity nucleotide site in the non-cycling enzyme, one whose affinity toward Co(NH3)4ATP seems to have experienced little change after FITC has covalently blocked the high affinity ATP site.

The Number of ATP Sites Per Protomeric Unit-- The above experiments support the view that two ATP-binding sites co-exist in Na,K-ATPase (6, 21, 27, 29-31). The controversial question has been whether this means that there is one high affinity and one low affinity ATP site per alpha beta protomer (27, 29, 30) or just a single site but within the framework of a membrane-bound (alpha beta )2 dimer whose halves work out of phase (47, 48). In the latter case, we have to assume that only one protomer in the dimer can bind and trap Co(NH3)4ATP, and that only this protomer loses all ATP responsiveness. One must further surmise that, through subunit interactions, the blocked protomer should cause its neighbor in the dimer to be confined to E1 conformations; the good protomer would not hydrolyze ATP but could be phosphorylated by it and catalyze ATP-ADP exchange. To test this model, we used C12E8 to solubilize (4-6) both Co(NH3)4ATP-modified and control Na,K-ATPase to alpha beta protomers. ATP-ADP exchange, K+ phosphatase, and Na,K-ATPase activities and protein were determined in membrane-bound and solubilized samples of inactivated and control enzymes. The specific activity (in µmol min-1 mg-1) was calculated for each reaction catalyzed by the protomers arising from the Co(NH3)4ATP enzyme and was expressed as percent left of the respective specific activity of the control protomers. Equivalent calculations were made with the parent, membrane-bound Co(NH3)4ATP-inactivated and control samples.

If the protomeric unit had one high affinity and one low affinity site and Co(NH3)4ATP only blocked the latter, the percent Na,K-ATPase activity left in the inactivated soluble protomers should be identical to that of the inactivated membrane-bound enzyme, and the same should apply to the K+ phosphatase activity. On the other hand, if single-site protomers were organized as an (alpha beta )2 dimeric membrane enzyme, and as C12E8 should have dissociated the enzyme to alpha beta protomers in our conditions (4-6), one would expect that the good but restrained protomer should now be rid of its inactive partner. Half of the Na,K-ATPase and K+ phosphatase activities that had been lost would now be restored. The results in Fig. 6 show that this is not so and agree with the 2-site option; the open plus solid bars illustrate the results expected from the single site plus dimer hypothesis. It is also evident that the ATP-ADP exchange activity of the Co(NH3)4ATP-treated enzyme remains high before and after solubilization. The experiment was repeated 12 times, with different preparations of purified enzyme and with variable Co(NH3)4ATP exposure, so as to obtain varying levels of inactivation; the results are shown in Fig. 7. The Na,K-ATPase and K+ phosphatase data conform rather well to the one-to-one correlation expected for two ATP sites per alpha beta protomer and are far removed from that predicted for the single site plus dimer hypothesis (upper dashed line). Table I presents the analytical ultracentrifugation results that show that solubilization of the Co(NH3)4ATP-modified enzyme with C12E8 does actually produce a protomeric preparation, just as it happens with the native (4-6), FITC-treated (49), or CrATP-treated enzymes (50). There was no difference in sedimentation coefficient between Co(NH3)4ATP-treated and control enzyme at any of the inactivation levels. The average s20,w returns a protein molecular mass of 158,000 ± 11,000, which is close to 147,500, the sum of alpha  and beta  proteins (2, 4-6, 29).


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Fig. 6.   The enzymic signature of Co(NH3)4ATP-modified Na,K-ATPase before and after solubilization to alpha beta protomers. Paired samples of native enzyme were incubated for 2.5 h at 37 °C in 20 mM imidazole (pH 6.3) with or without 1 mM Co(NH3)4ATP. The samples were centrifuged, washed, and resuspended with 300 mM NaCl, 40 mM TES (pH 7.4), and their protein concentrations were determined and adjusted to 680 µg ml-1. The Na,K-ATPase activity was assayed in 1:20 enzyme dilutions of the control and Co(NH3)4ATP-modified enzymes, as described under "Experimental Procedures" (except that 10 mM TES (pH 7.5) replaced the imidazole); the K+ phosphatase and ATP-ADP exchange activities were determined likewise (with 10 mM TES, pH 7.5, and using 20 mM pNPP for the former and 150 mM NaCl for the latter). The specific activities of the modified membrane-bound enzyme (MB, cross-hatched bars) are shown as percent of the respective specific activities of the control enzyme. Aliquots of the modified and control enzymes were diluted 1:1 with C12E8 (2 mg ml-1) to obtain soluble alpha beta protomers, and protein and enzymic activities were now determined in the 425,000 × g supernatants. The specific activities of the protomeric Co(NH3)4ATP-enzyme are shown (alpha beta , solid bars) as percent of the specific activities of the control protomeric enzyme. The open plus solid bars represent percent specific activities that would be expected for the soluble protomer if the membrane-bound enzyme consisted of (alpha beta )2 dimers with a single ATP site per alpha beta protomeric unit. Vertical lines are compounded errors of the mean.


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Fig. 7.   Correlation between the Na,K-ATPase and K+ phosphatase activities of Co(NH3)4ATP-modified, membrane-bound sodium pump and the respective activities of the resulting soluble protomers. Paired samples of membrane-bound Na,K-ATPase were incubated with and without Co(NH3)4ATP as in Fig. 6, in different experiments, so as to obtain various degrees of inactivation. The protein concentrations and enzymic activities were determined in the Co(NH3)4ATP-modified enzyme and in the parallel control before and after solubilization to alpha beta protomers. The specific activities of soluble and (parent) membrane-bound inactivated enzymes have been expressed as in Fig. 6 and plotted against each other. Solid symbols, Na,K-ATPase activity; open symbols, K+ phosphatase activity. Bars show the compounded errors. Dashed and dotted lines represent expectations for the hypotheses tested; for explanation, see main text.


                              
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Table I
Analytical ultracentrifugation of Co(NH3)4ATP-modified Na,K-ATPase and native Na,K-ATPase after solubilization with C12E8
Co(NH3)4ATP-treated Na,K-ATPase was solubilized with C12E8, side by side with a parallel control (see "Experimental Procedures"). Sedimentation velocity analysis of the 435,000 × g supernatants was carried out in a Beckman Optima XL-A analytical ultracentrifuge. The inactivated and control solubilized enzymes were analyzed in the same run, using double-sectored centrepieces, at 40,000 rpm. and 20 °C. Single sedimentation fronts were found in all cases, and the sedimentation coefficients (s20,w) and their errors were determined as described under "Experimental Procedures." Using a Stokes radius of 72 Å and a partial molar volume = 0.646 cm3 g-1(4), the calculated protein molecular mass works out as 158,000 ± 11,000. The specific Na,K-ATPase activities were determined in samples of the inactivated and parallel control enzymes; the former is shown as percent of the latter.

Direct Co(NH3)4ATP Inactivation of Solubilized alpha beta Protomers-- Because of the limited thermal stability of the soluble enzyme (49), we first tried lowering the temperature to 20 °C to demonstrate direct Co(NH3)4ATP inactivation of the soluble protomers over periods similar to those in Fig. 2. As this was unsuccessful, we decided to take advantage of the greater thermal stability of the soluble alpha beta protomer at 37 °C when in the presence of low K+ concentrations, in Na+-free, Mg2+-free medium (49). Fig. 8A (open circles) shows that the soluble protomers were quite stable at 37 °C in the presence of 10 mM K+. This panel also shows that 50 µM Co(NH3)4ATP was exceedingly effective under these conditions, causing enzyme inactivation at a rate much faster than obtained with the membrane-bound enzyme at higher analogue concentrations (compare with Fig. 2). When ATP was included in the medium, the inactivation rate decreased to less than half. The experiment in Fig. 8B shows the Co(NH3)4ATP concentration dependence of the inactivation process. In this case, the rate constant kinact has been estimated from a single time point (2.5 min) under the assumption of single exponential decay, and this can only be an approximation. It seems unlikely, however, that this degree of uncertainty could explain the marked differences with respect to the kinetics of inactivation of the membrane-bound enzyme (Fig. 3A). Relative to the latter, the maximal inactivation rate of the soluble protomers was now 12 times faster, and the affinity nearly 40-fold higher during the Co(NH3)4ATP binding event that precedes irreversible enzyme modification.


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Fig. 8.   Direct Co(NH3)4ATP inactivation of soluble protomeric Na,K-ATPase. The native enzyme was solubilized with C12E8 in a medium containing 10 mM KCl, 20 mM TES (to pH 7.25 with triethanolamine). The K+ phosphatase activity was measured in 1:6 dilutions of the high speed supernatant, as linear time courses over 8 min at 20 °C. A, soluble protomers were incubated at 37 °C; samples were stopped by dilution and cooling at the times shown, and their K+ phosphatase activity was determined. Three separate runs, with (i) no additions (open circles), (ii) 50 µM Co(NH3)4ATP (solid circles), and (iii) 50 µM Co(NH3)4ATP plus 2 mM ATP (open triangles) or without ATP (solid triangles). Vertical lines show the S.E. B, soluble protomers were preincubated for 2.5 min at 37 °C, at the Co(NH3)4ATP shown, and their K+ phosphatase activity was determined as above. An estimate of the inactivation rate constant kinact has been plotted on the ordinate; the data have been fitted to the hyperbola shown, with KD = 16 ± 1 µM and kinact(max) = 28.8 ± 0.6 h-1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Co(NH3)4ATP and FITC as ATP Site Probes-- As a first step, we confirmed that it was possible to measure ATP-[3H]ADP exchange following low affinity Co(NH3)4ATP inactivation of E2 functions and turnover (Fig. 2) (32). Although the dephosphorylating activity had been blocked, the minimal enzyme unit retained functional phosphorylation and high affinity nucleotide sites, as well as the catalytic machinery and conformational freedom needed for reversible kinase activity.

The results in Fig. 3 show that high and low affinity Co(NH3)4ATP binding are properties inherent to the native non-cycling Na,K-ATPase. Complexation of ATP by the Co(III)(NH3)4 group does not lead to deleterious effects on high affinity binding to the enzyme (Fig. 3B), and it might even lower KD values. It is also clear, and despite suggestions to the contrary (51), that low affinity nucleotide binding (Fig. 3A) is not the result of previous FITC modification nor the kinetic outcome of a branched reaction cycle. All the same, Fig. 5 shows that Co(NH3)4ATP can also bind to the FITC-modified enzyme, with low affinity and without hindrance. The ensuing Co(NH3)4ATP trapping inactivates the K+ phosphatase activity that had survived the FITC treatment and not only does the low Co(NH3)4ATP binding affinity remain unaltered, but there is also little change in the maximal inactivation rate constant. Evidently, the occluded Co(NH3)4ATP state is being reached with a probability similar to that in the native enzyme, and this suggests that the conformational flexibility around this extant low affinity site is preserved after FITC modification of the enzyme.

Two Sites Per Protomer or a Dimeric Membrane Enzyme?-- If there were a single ATP site per alpha beta protomer, to explain the unfaltering Co(NH3)4ATP inactivation of the FITC-modified membrane enzyme (Fig. 5), we would need to postulate that the minimal functional unit of Na,K-ATPase was an (alpha beta )2 membrane dimer, at the very least. The Co(NH3)4ATP inactivation pattern observed with the native membrane-bound enzyme could then be explained if the analogue was trapped by only one-half of the hypothetical dimer. This would lock the other half in an E1 conformation (32) and allow substantial survival of the ATP-ADP exchange activity while causing total loss of Na,K-ATPase activity. We applied the solubilization test used earlier (29) which showed that FITC could access and inactivate the high affinity ATP site of every alpha beta protomer in the membrane-bound enzyme. In the present case, the expectation with respect to the Co(NH3)4ATP-modified enzyme was that solubilization to single protomers would increase the Na,K-ATPase activity if the putative membrane-bound (alpha beta )2 dimer had one good and one Co(NH3)4ATP-blocked protomer, with one ATP site each protomer. The experiments in Figs. 6 and 7 show that this was not the case. At face value, the conclusion must that both ATP sites should be found on every alpha beta protomer.

The demonstration above rests on the assumption that the enzymic reactions catalyzed by the solubilized enzyme genuinely represent the activity of the alpha beta protomer. Table I shows that the sedimentation coefficients obtained for the Co(NH3)4ATP-modified and native enzymes were identical and returned, within error, the protein molecular mass of the alpha beta particle (4-6). By applying the method of active enzyme ultracentrifugation, we had shown earlier (6) that in our solubilization conditions (weight ratio C12E8/protein = 3) the alpha beta protomer was the only soluble species with Na,K-ATPase activity. We then also found that the dual nucleotide effects on the activity were inherent to the soluble protomeric enzyme (6), i.e. there was no need to advocate a membrane (alpha beta )2 dimer to explain this basic feature. However, our results are still at odds with those obtained by high performance gel filtration/laser light scattering of the C12E8-solubilized enzyme (52, 53), which shows a substantial level of (alpha beta )2 dimers in equilibrium with the alpha beta protomer. Although we do not have an explanation for the discrepancy, we have pointed out (6, 29) that, in the event of such equilibrium, our s20,w measurements always return an upper estimate of the weighted-average molecular mass of protomer and putative dimer (54), because of dimer dissociation upon dilution. The steady-state properties of the gel filtration protomers and dimers have not been assessed (52, 53) but, even using KA = 5 × 105 M-1 at 20 °C (52), at a protein concentration <= 1.9 × 10-7 M (Figs. 6-8), the calculated contents of soluble dimers works out as <7%.

Another explanation has been suggested (48) for results like those in Figs. 6 and 7, that a very small dimer population that would escape detection during analytical ultracentrifugation could exist in rapid equilibrium with the protomer. That dimer, and not the protomer, would be the only soluble particle with enzymic activity or would at least be the source of non-Michaelian ATP effects. Apart from being kinetically unlikely (6), this possibility is excluded by the very same results in Fig. 7 (also see Fig. 3 of Ref. 29). The reason, again, is that if only one alpha beta protomer in the (alpha beta )2 membrane dimer is blocked directly by Co(NH3)4ATP (or FITC for that matter), total loss of Na,K-ATPase activity can only result from restrictive subunit interactions within the dimer. But then as protomer and dimer will be in rapid equilibrium after solubilization, one can safely predict that good and bad alpha beta protomers will be instantly randomized within the small soluble (alpha beta )2 population. For instance, if the E2 partial reactions and the Na,K-ATPase activity of the membrane enzyme are 100% inactivated, the outcome of solubilization/recombination will be 50% hybrid dimers, 25% twice-inactivated dimers, and 25% intact dimers, i.e. the reshuffle shall give away the presence of the stealthy dimer. Let p be the probability of intact alpha beta solubilized from membrane (alpha beta )2 at each inactivation level, i.e. p = 0.5 at zero membrane activity, 0.75 at 50% membrane activity, etc. The probability of forming active soluble dimers is then p2 which, expressed as percent, is shown by the dotted line in Fig. 7. As only the good soluble (alpha beta )2 shall be active, the relative specific activity of the soluble enzyme must be equal to the ratio (good soluble dimer concentration per mg of inactivated enzyme)/(good soluble dimer concentration per mg of control enzyme). From the basic expression for a protomer/dimer equilibrium it is easily shown that, at low dimer levels (i.e. the condition for the "concealed dimer" proposal (48)), the above ratio is very approximately p2. This is because we should expect a unique association constant KA for the dimerization of intact soluble alpha beta , whether it arises from the inactivated or the control enzyme. Therefore, the dotted line in Fig. 7 also represents the predicted correlation for the relative specific activity of the soluble enzyme, expressed as percent. That was not the correlation for either Co(NH3)4ATP (Fig. 7) or FITC (Fig. 3 in Ref. 29). The option of an active soluble dimer in fast equilibrium with an inactive protomer (48) may, therefore, be discarded.

Direct Co(NH3)4ATP Inactivation of the Soluble alpha beta Protomer-- By incubating at pH 7.25 and 37 °C, we could demonstrate Co(NH3)4ATP-dependent inactivation of more than 90% of the solubilized enzyme (Fig. 8A), despite the presence of K+ (31). This shows that all alpha beta protomers are intrinsically and equally susceptible to the analogue and reinforces the results of Figs. 6 and 7. An interesting feature is the ~40-fold increase in Co(NH3)4ATP binding affinity, KD = 16 µM (Fig. 8B), by comparison with the membrane-bound enzyme, KD = 0.41-0.62 mM (Figs. 3A and 5C). This lower KD is as would be expected from the ATP concentration dependence of the Na,K-ATPase activity of the soluble protomer at 20 °C (6), where the low affinity K0.5 was 21 µM (as opposed to 175 µM for the membrane-bound enzyme). The maximal turnover rates of membrane-bound and solubilized enzymes are very similar, in conditions such that low affinity ATP binding controls the rate-limiting step (4-6). This suggests that the lower K0.5 reflects a decrease in real dissociation constant (KD koff/kon) at the low affinity site. It is perhaps a lower koff that better explains this change, as a 10-fold faster Co(NH3)4ATP inactivation rate constant (kinact) was observed with the soluble protomer (Figs. 3A and 8B). A slower off-rate will result in a longer lifetime for the initial [enzyme·Co(NH3)4ATP] complex in detergent solution; the complex should thus have the opportunity to sample a greater number of sub-conformational states, with a higher probability of finding that conducive to Co(NH3)4ATP trapping and site inactivation. Of course, the higher ATP and Co(NH3)4ATP affinities in C12E8 solution may be the result of changes in protein conformation caused by detergent binding and the formation of protein micelles. It is possible that the phase transitions independently increase the rate of Co(NH3)4ATP inactivation as well.

Evidence Against Two ATP Sites-- In a study with enzyme purified from duck nasal gland (51), the Na,K-ATPase and K+ phosphatase activities appeared to be equally inhibited by TNP-ADP, or inactivated by increasing ErITC concentrations. However, the ErITC data should be interpreted with caution as the inactivation was evaluated only after stoichiometric completion had been reached, and this may have concealed different ErITC concentration dependences of the rate constants (38). At any rate, in the case of SERCA (55) ErITC inactivates the low affinity ATP effect on the Ca-ATPase reaction far more effectively than the high affinity effect. As the behavior of the K+ phosphatase activity tends to line up with low affinity ATP interactions and the Na,K-ATPase activity was only measured at high ATP concentrations (51), the finding with the sodium pump might not be addressing the relevant issue. On the other hand, with gastric H,K-ATPase (56) there is an apparent and compound dilemma that TNP-ATP binds with a single affinity but a stoichiometry of 2 mol per phosphorylation site and that ATP competes the TNP-ATP binding with two ATP dissociation constants differing by a factor of >300. This illustrates the risk that even real affinities can be misleading if used selectively and, in isolation, as ultimate evidence (a caveat that cuts both ways, as discussed by Faller (56)).

A recent study (57) has reported results of site-directed mutagenesis of residues lining a predicted nucleotide binding pocket in rat alpha 1-Na,K-ATPase, by assimilation to the N domain of SERCA (24). Mutants were expressed in HeLa cells and selected by virtue of their ouabain resistance; none of the mutations drastically affected low affinity ATP binding. The difficulty with this approach is that the mutants sought could not have been viable, because their Na+ pumping rate would have been less than 5% normal. Because the endogenous pump was inhibited by ouabain, the rise of intracellular Na+ would have caused colloid-osmotic swelling and lysis of the host cells. Despite the selection procedure, it could be found that in the case of R544A there was a 59-fold increase in K0.5 for phosphorylation by ATP and only a 2-fold increase in K0.5 for the low affinity ATP effect (57). This shows that it is at least possible to interfere with high affinity ATP binding with little effect on low affinity binding.

The evidence against the existence of a regulatory ATP site is broadly based on experiments that show that although binding at an apparently single site, some ligands can inhibit more than one Na,K-ATPase function or interfere with both high and low affinity ATP effects (51, 57, 58). Although these findings are interesting in their own right, the possibility should be considered that some of these agents or procedures act through global effects, the case of ouabain is a prime example, or that the experimental conditions are unfavorable for the question at hand (27, 58). In fact, the converse remains the crucial quandary, i.e. that certain ATP analogues can selectively inhibit or inactivate some partial reactions and not others or do so with different affinities. For instance, Cr(H2O)4-adenosine 5'-[beta gamma -methylene]triphosphate (59) inactivates ATP phosphorylation with a high affinity but does not affect E2 reactions, i.e. it has effects opposite to those of Co(NH3)4ATP. Among agents that bind covalently to Na,K-ATPase, p-fluorosulfonylbenzoyl-adenosine (60), 8N3-ATP (27), 2N3-ATP (61), and FITC (25, 29) are, at low concentrations, far more effective at inactivating the Na,K-ATPase than the K+ phosphatase activity. These affinity labels become incorporated in the alpha  subunit at Lys719, Lys480, Gly502, and Lys501, respectively (22, 62-64).

ATP Sites and the Tridimensional SERCA Structure-- On balance, the results of this work support the notion of independent high and low affinity ATP-binding sites in the protomeric unit. When considering whether FITC and Co(NH3)4ATP might bind within one broad nucleotide site or at two separate pockets, the crucial point is that if the former is true, docking of these ligands at their locations should be non-overlapping and apparently non-interacting. Recent preliminary experiments measuring fluorescence energy transfer return a Förster distance of 29 Å between FITC and the cobalt of Co(NH3)4ATP bound at equilibrium to Na,K-ATPase (65, 66). In the high resolution open SERCA structure (24), this is roughly the distance between Lys515, the anchoring point of FITC in the N domain, and Asp351, the phosphorylation site on the P domain, and by far exceeds the span of an ATP molecule. A single nucleotide-binding site has been located in the N domain (24) from the increased electron density obtained after diffusion of TNP-AMP into the crystals. TNP-AMP is the nucleotide with the highest affinity toward SERCA, and TNP-[3H]AMP binds at equilibrium with a stoichiometry of 1 mol per phosphorylation site (67). However, although 2 mol of TNP-[3H]ATP can be bound per mol of SERCA in similar conditions, and two competing ATP effects may be observed (67, 68), there have been no reports of a second ATP site in SERCA crystals. As mentioned above, twice stoichiometric TNP-ATP binding and dual ATP competition can also be seen with gastric H,K-ATPase (56).

Recent experiments (69, 70) have shown that TNP-8N3[alpha -32P]ADP photolabels alpha Lys480 of native Na,K-ATPase, i.e. the same residue as 8N3-ATP (63); the equivalent Lys492 is the TNP-8N3-ATP target in the N domain of SERCA (71). But then close to 1 mol of TNP-8N3-[alpha -32P]ADP can be incorporated per mol of FITC-modified alpha Na,K-ATPase, with a lower affinity (30), now in the tryptic hexadecapeptide beginning at alpha Ala721 of the P domain (69, 70). In the open SERCA structure (24), Lys492 is 25-30 Å away from the fragment equivalent to alpha Ala721-Lys736 in Na,K-ATPase; this distance is similar to that between FITC and Co(NH3)4ATP in the sodium pump (65, 66). The P domain might then be one of the platforms accommodating a low affinity ATP-binding site. This might also be the case with the site for the phosphatase substrate, as 4N3-2-nitrophenyl phosphate can photolabel Na,K-ATPase at alpha Pro668 and alpha Asn398 (72), presumably in its P and N domains, respectively.

    ACKNOWLEDGEMENTS

We thank Prof. W. Schoner (Justus-Liebig Universität, Giessen, Germany) for samples of Co(NH3)4ATP and advice on its synthesis and use, and Prof. A. Rowe, National Centre for Macromolecular Hydrodynamics, for help with the analytical ultracentrifugation.

    FOOTNOTES

* This work was supported by research grants from The Wellcome Trust and the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

§ To whom correspondence should be addressed. Tel./Fax: 44-116- 2523091; E-mail: jdc7@le.ac.uk.

Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211128200

    ABBREVIATIONS

The abbreviations used are: Na, K-ATPase, (Na+ + K+)-activated adenosine triphosphatase; Co(NH3)4ATP, Co(III) tetrammine ATP; Co(NH3)4PO4, Co(III) tetrammine phosphate; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; C12E8, octaethylene glycol dodecyl monoether; pNPP, p-nitrophenyl phosphate; 3-OMFP, 3-O-methylfluorescein phosphate; 3-OMF, 3-O-methylfluorescein; FITC, fluorescein 5'-isothiocyanate; TNP-AMP, -ADP, or -ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5' mono-, di-, or triphosphate; 2- or 8N3-ATP, 2- or 8-azido-adenosine 5'-triphosphate; TNP-8N3-ADP, 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate; ErITC, erythrosin 5'-isothiocyanate; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Glynn, I. M. (1985) in The Enzymes of Biological Membranes (Martonosi, A. N., ed), Vol. 3 , pp. 35-114, Plenum Publishing Corp., New York
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