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J Biol Chem, Vol. 273, Issue 50, 33759-33765, December 11, 1998

Affinity Labeling of Two Nucleotide Sites on Na,K-ATPase Using 2'(3')-O-(2,4,6-Trinitrophenyl)8-azidoadenosine 5'-[-³²P]Diphosphate (TNP-8N₃-[-³²P]ADP) as a Photoactivatable Probe
LABEL INCORPORATION BEFORE AND AFTER BLOCKING THE HIGH AFFINITY ATP SITE WITH FLUORESCEIN ISOTHIOCYANATE^*

Douglas G. Ward and José D. Cavieres

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

	ABSTRACT

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ATP and its analogues act on the minimal functional unit of Na,K-ATPase, the protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K⁺-phosphatase activity of soluble FITC-modified protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N₃-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N₃-[-³²P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 µM TNP-8N₃-[-³²P]ADP, and ATP or FITC treatment prevents labeling of the  chain. At 25 µM, however, TNP-8N₃-[-³²P]ADP can be incorporated in the FITC-modified  chain, concurrently with the inactivation of the K⁺-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of ³²P-probe per mol of  chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na⁺ or Mg²⁺, but not K⁺. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N₃-[-³²P]ADP·Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na⁺ site. Partial trypsinolysis reveals that the point of TNP-8N₃-[-³²P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified  chain. The affinity labeling of the fluorescein enzyme by TNP-8N₃-[-³²P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each  subunit of Na,K-ATPase.

	INTRODUCTION

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Na,K-ATPase¹ is the integral plasma-membrane enzyme that catalyzes Na⁺ and K⁺ transport as it hydrolyzes ATP. The sodium pump consists of a 112-kDa  subunit (1, 2) and one  subunit, 35 kDa in protein and 50-60 kDa after glycosylation (3). In the membrane, the enzyme may be organized into ()₂ dimers (4) or higher oligomers; however, solubilized protomers hydrolyze ATP and catalyze many of the partial reactions associated with the pumping cycle (5, 6). The phosphorylation, ouabain, cation, and high affinity ATP binding sites are found on the  subunit (7). The  subunit is required for correct membrane insertion (8) and may play a role in K⁺ transport and in stabilizing the  subunit (9, 10).

In the presence of K⁺, the activity of Na,K-ATPase exhibits a complex non-Michaelian dependence on ATP concentration (11). The requirement for steady-state phosphorylation is satisfied at micromolar ATP, and yet the rates of hydrolysis and cation transport increase 20-50-fold if ATP is raised to millimolar levels (12); this clearly requires more than one encounter between ATP and the enzyme during the reaction cycle. Thus far, it had seemed economical to attribute the apparent multiplicity of ATP binding sites to the structure, probably oligomeric, of the membrane-bound enzyme. This hypothesis entails cooperativity between protomers, with each  subunit having an unique and structurally identical (but conformationally different) ATP binding site. It has also been proposed that an enzyme with a single site, which would alternate between high and low-affinity conformations, could account for the complex behavior with respect to ATP (13). This seemed a plausible alternative when the C₁₂E₈ (octaethylene glycol dodecyl monoether)-solubilized protomers were shown to present dual responses to ATP in the absence of oligomerization (6).

Our current work supports yet a third possibility: that each protomer presents separate high and low affinity ATP sites. We found that when FITC-modified Na,K-ATPase was solubilized with C₁₂E₈, the soluble protomers retained their K⁺-phosphatase activity but did not recover their ability to hydrolyze ATP (14); it follows that FITC must effectively block the high affinity ATP site of every protomer and not just one half of an ()₂ membrane dimer. The K⁺-phosphatase activity of these protomers could be inhibited by TNP-ADP (14), a tight-binding ATP analogue (15), which was more effective than TNP-UDP (16); it could also be inhibited by the azido derivative TNP-8N₃-ADP. Upon UV irradiation, TNP-8N₃-ADP inactivated both pNPPase activity and P_i phosphorylation in a process that was accelerated by Na⁺ or Mg²⁺ and inhibited by TNP-ADP or K⁺. TNP-8N₃-ADP also photoinactivated the FITC-modified, solubilized protomers (16). The simultaneous covalent binding of FITC and TNP nucleotide to the same soluble protomer suggested that high and low affinity ATP binding occurred at two distinct sites on the protomeric enzyme unit.

Previously, we had used indirect methods to demonstrate TNP-nucleotide binding to the fluorescein enzyme (14, 16). In this paper, we describe the use of TNP-8N₃-[-³²P]ADP, first as an affinity label for the high-affinity (catalytic) ATP site; we go on to show directly that the FITC-modified  subunit can covalently bind TNP-8N₃-[-³²P]ADP. It seems likely, therefore, that the site of primary TNP-8N₃-[-³²P]ADP attachment to the FITC-modified Na,K-ATPase is within a regulatory nucleotide pocket distinct from the catalytic ATP binding site.

	EXPERIMENTAL PROCEDURES

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Enzyme Purification-- Na,K-ATPase was purified from pig outer renal medulla with the zonal rotor method of Jørgensen (17). The Na,K-ATPase activity ranged from 20 to 35 units/mg of protein at 37 °C.

Na,K-ATPase Assay-- Na,K-ATPase activity was measured using a coupled enzyme spectrophotometric assay at 37 °C, as described previously (16).

K⁺-Phosphatase Assay-- The pNPPase activity was determined from the linear time courses of p-nitrophenol release at 20 °C (14). Assay conditions were as follows: 20 mM KCl, 20 mM Tris-Cl (pH 7.5), 6 mM MgCl₂, 1 mM EDTA, and 10 mM pNPP.

FITC Treatment-- Membrane-bound purified Na,K-ATPase was suspended at 1 mg of protein/ml in 150 mM NaCl, 50 mM Tris (pH 9.2), and 5 mM EDTA. FITC was prepared as a 10 mM stock solution in dimethyl sulfoxide and added to give a final concentration of 50 µM. The suspension was incubated at 20 °C for 30 min in the dark; the membranes were spun down and washed in a Beckman TL-100 ultracentrifuge, and the pellet was homogenized in 20 mM Tris-Cl (pH 7.5), 1 mM EDTA. A control enzyme sample was processed side by side, with the omission of FITC. In all cases, the FITC-treated enzyme retained less than 1% of the Na,K-ATPase activity of the parallel control.

8N₃-[-³²P]ADP Preparation-- Two different approaches were used to obtain 8N₃-[-³²P]ADP. 1) 8N₃-[-³²P]ATP was purchased and hydrolyzed to 8N₃-[-³²P]ADP with Na,K-ATPase. Analytical high performance liquid chromatography on a strong anion exchange column confirmed that hydrolysis was complete. 2) 8N₃-[-³²P]ADP was synthesized from [-³²P]ATP (18). This was converted to 8Br-[-³²P]ATP by mixing 3 µmol of [-³²P]ATP (9 MBq) dissolved in 250 µl of 1 M sodium acetate/acetic acid buffer (pH 3.8) with 250 µl of bromine water (5 µl of Br₂/ml). The reaction was essentially complete after 4 h at room temperature. The 8Br-[-³²P]ATP was purified on a DEAE-Sephadex column (1.6 × 20 cm) using a gradient of 0-1 M triethylammonium formate (pH 5.3). The 8Br-[-³²P]ATP peak was collected, lyophilized, co-evaporated four times with methanol, and then dissolved in 10 ml of dimethylformamide containing 10 mmol of triethylamine azide, prepared according to Haley (19). The reaction mixture was heated to 75 °C for 10 h. The dimethylformamide was evaporated to a syrup, and the 8N₃-[-³²P]ATP purified by a repeat of the DEAE-Sephadex chromatography. The final stage was to convert the 8N₃-[-³²P]ATP to 8N₃-[-³²P]ADP, using Na,K-ATPase.

TNP-8N₃-[-³²P]ADP Synthesis-- TNP group addition to 8N₃-[-³²P]ADP was according to the method of Seebregts and McIntosh (20), modified as described previously (16). The product was purified by reverse phase high performance liquid chromatography on a C₁₈ column (250 × 4.6 mm, Hichrom). A linear gradient (0-100% B) was delivered over 60 min (A = 50 mM triethylammonium formate, pH 6.0; B = methanol), and the peak containing TNP-8N₃-[-³²P]ADP was collected, lyophilized, dissolved in 20 mM Tris (pH 7.5), 1 mM EDTA, and stored at 80 °C. The yield was approximately 50%. The product had the correct UV/visible absorption spectrum (20) and migrated as a single peak when reapplied to the C₁₈ column.

Photoinactivation-- The enzyme was suspended in 20 mM Tris (pH 7.5), with extra additions as specified in the legends to figures, and pipetted into multiwell dishes to a depth of 2-3 mm. The dishes were placed inside a chamber at room temperature and constant humidity and irradiated with a Flowgen VL-6MC UV lamp set at 312 nm, as described (16).

Electrophoresis-- SDS-polyacrylamide gel electrophoresis of unboiled Na,K-ATPase samples (21) was carried out according to Laemmli (22). The samples were run on 3% T stacking and 10% T separating slab gels in a Hoefer minigel apparatus at a constant voltage (150 V). All gels were stained with Coomassie Brilliant Blue R-250 and destained with 7% acetic acid, 45% methanol. For autoradiography, the gels were dried, and the x-ray film was exposed with an intensifying screen for 8-72 h at 80 °C.

Electroelution-- Bands were cut from Coomassie-stained SDS-polyacrylamide gels and eluted at 10 mA per tube for 6-8 h at room temperature, using a Bio-Rad model 422 electroeluter according to the manufacturer's instructions. Protein concentration and radioactivity were determined in aliquots of the nondialyzable eluate against appropriate standards, and the number of nmol of TNP-8N₃-[-³²P]ADP per mg of  chain protein were calculated.

Controlled Trypsinolysis-- FITC-modified Na,K-ATPase was photolabeled with TNP-8N₃-[-³²P]ADP, spun down, and washed in the benchtop ultracentrifuge. The labeled enzyme was resuspended in a medium containing 20 mM Tris-Cl (pH 7.5), 1 mM EDTA and incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (2 µg per 90 µg Na,K-ATPase) at 37 °C. The trypsin action was stopped in aliquots, at various times, with a 2.5-fold mass excess of soybean trypsin inhibitor. Samples were drawn and mixed with sample buffer (22) for SDS-polyacrylamide gel electrophoresis.

Protein Assays-- Protein was precipitated with 7% trichloroacetic acid and quantitated with the bicinchonninic acid method (23) as described previously (14).

Curve Fitting and Errors-- All enzyme activities were calculated from the regression coefficients of least-squares linear regressions on product release time-courses. Error bars in Figs. 3 and 5 represent the S.E. on multiple determinations, compounded where appropriate. The photoinactivation time-courses did not conform to simple (single or double) exponential decays (16); single exponential functions with a nonzero infinity value could be fitted by nonlinear regression using SigmaPlot 4.0.

Materials-- ATP (disodium salt) was from Boehringer Mannheim; FITC and TNP-ADP were from Molecular Probes; 8N₃-ADP, 8N₃-ATP, 2,4,6-trinitrobenzenesulfonic acid, pNPP (Tris salt), trypsin inhibitor, DEAE-Sephadex A-25-120, molecular mass markers, and other materials for electrophoresis and Na,K-ATPase assays were from Sigma. [-³²P]ATP was from NEN Life Science Products, and 8N₃-[-³²P]ATP was from ICN. Dimethyl sulfoxide, 5,5'-dithiobis(2-nitrobenzoic) acid, and trifluoroacetic acid were from Aldrich. Blue-Sensitive x-ray film was from Genetic Research Instrumentation. Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) was from Worthington. All other reagents were of the highest purity available.

	RESULTS

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In order to evaluate the specificity of TNP-8N₃-[-³²P]ADP toward ATP sites in Na,K-ATPase, we performed initial experiments with the native enzyme. We had recently shown that TNP-8N₃-ADP photoinactivated the Na,K-ATPase activity of the native sodium pump and that that the enzyme could be protected by excess TNP-ADP. In those experiments, at 5 µM TNP-8N₃-ADP, about 30% of the Na,K-ATPase activity remained after 25 min of UV irradiation (16). The results in Fig. 1 show that when used with the same inactivation protocol, TNP-8N₃-[-³²P]ADP is incorporated in the  subunit of the native enzyme. Under these conditions, 100 mM Na⁺ (Fig. 1, lane 1) or 10 mM Mg²⁺ (lanes 6 and 7) has little or no effect on the labeling obtained with just 20 mM Tris-Cl (lane 2), whereas 100 mM K⁺ (lane 3) causes a somewhat lower level of incorporation. The K⁺ effect is indeed expected as it decreases the nucleotide binding affinity at the catalytic site (24, 25). The photolabeling can be prevented by including 2 mM ATP (lane 4) or 0.1 mM TNP-ADP (lane 5) in the medium. Likewise, covalent modification of the enzyme with FITC (lane 8) impedes access of TNP-8N₃-[-³²P]ADP at this concentration of the probe (5 µM). Considering that ATP protects the  chain and that FITC blocks a high-affinity ATP site (16, 26), it is apparent that the trinitrophenyl group, which increases binding affinity (15), does not have much effect on binding specificity (cf. Refs. 14 and 16). It is most likely, therefore, that the TNP-8N₃[-³²P]ADP labeling in Fig. 1 occurs from within the catalytic ATP site of native Na,K-ATPase.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   TNP-8N3-[-32P]ADP photolabeling of native Na,K-ATPase. Aliquots of Na,K-ATPase were photolabeled with 5 µM TNP-8N3-[-32P]ADP for 30 min, in the presence of 20 mM Tris-Cl (pH 7.5), 1 mM EDTA, and the additions shown below. The samples were spun down in the benchtop ultracentrifuge and washed with and resuspended in 20 mM Tris-Cl (pH 7.5). The protein concentration was determined, and lanes 1-8 were loaded with 4 µg of protein. Top panel, Coomassie stain; left lane, molecular mass markers. Lower panel: autoradiogram. Photolabeling conditions were 100 mM NaCl (lane 1), no additions (lane 2), 100 mM KCl (lane 3), 100 mM NaCl + 2 mM ATP (lane 4), 100 mM NaCl + 100 µM TNP-ADP (lane 5), 10 mM MgCl2 (lane 6), and 100 mM NaCl + 10 mM MgCl2 (lane 7). A sample of FITC-modified Na,K-ATPase was also subjected to the same labeling procedure, in the presence of 100 mM NaCl, and 4 µg were loaded in lane 8. The arrows indicate the position of  and  chains, of the molecular mass markers (mass in kDa), and of the gel front (F).

The FITC-modified sodium pump cannot catalyze E1 partial reactions or the overall (i.e. Na,K-ATPase) reaction. However, E2 functions, such as K⁺-phosphatase and phosphorylation by inorganic phosphate, are more or less unaffected (14, 16, 26-29). The E2 partial reactions can still be reversibly inhibited by ATP analogues, such as TNP-ADP, acting with low affinity on the membrane-bound fluorescein enzyme (28, 29) and, crucially, on the soluble protomers prepared from it (14). The surviving pNPPase activity and [³²P]P_i phosphorylation of the FITC enzyme can be photoinactivated using TNP-8N₃-ADP at concentrations higher than those required to inactivate the Na,K-ATPase activity of the native enzyme (16), and this lends support to the idea of a distinct low-affinity nucleotide site on the protomer. It had been suggested (28) that the selective survival of E2 functions could be due to a rotation of the fluorescein molecule about its tether, so that access to the active site was possible in the E2 (K⁺) but not in the E1 (Na⁺) conformer. In our former study, we found that the TNP-8N₃-ADP photoinactivation of the fluorescein enzyme was accelerated by Na⁺ or Mg²⁺, and that K⁺ counteracted their effect. We argued that these effects were exactly the opposite as predicted for TNP-8N₃-[-³²P]ADP access to the blocked site and that this made the hypothesis of fluorescein rotation very unlikely (16). These cation effects are now expanded in the experiments shown in Fig. 2 (this and subsequent figures refer to the FITC-treated enzyme). Note that the photoinactivation of the pNPPase activity of the FITC-modified sodium pump is conducted at a TNP-8N₃-ADP concentration five times higher than that used in the experiments with the native enzyme (Fig. 1). The ordinate is intended to approximate the format of the inactivation rate constant, which would have been calculated had the time courses been simple exponentials. The MgCl₂ and NaCl effects occur with low affinities, and MgCl₂ promotes a greater maximal inactivation (63% versus 54%). The MgCl₂ effect correlates reasonably well with the calculated level of TNP-8N₃-ADP·Mg complex (Fig. 2B, dotted curve). The hyperbolic dependence on the Na⁺ concentration (Fig. 2A) might mean that a single Na⁺ site is involved, and the high apparent K_0.5 (97 mM) suggests an "extracellular" effect (30-34). This is reminiscent of the allosteric external Na⁺ site (32).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   The effects of sodium (A), magnesium (B), and potassium (C) on the TNP-8N3-ADP photoinactivation of FITC-modified Na,K-ATPase. FITC-modified Na,K-ATPase was suspended in 20 mM Tris (pH 7.5), 25 µM TNP-8N3-ADP plus varying amounts of NaCl, MgCl2 or KCl (the latter in the presence of 10 mM MgCl2), as indicated on the abscissa. The pNPPase activity was determined in a standard medium (see under "Experimental Procedures") in triplicate, before and after 30 min of UV irradiation. The negative value of the natural logarithm of the fraction remaining is plotted on the ordinate. Hyperbolae have been fitted to the cation-dependent components by nonlinear regression. The fitted parameters (K0.5 and maximal percentage of activity remaining) are as follows: A (Na+), 97 ± 9 mM and 46%; B (Mg2+), 1.3 ± 0.3 mM and 37%; and C (K+), 2.5 ± 0.6 mM and 72% (from 42% with no K+). The dotted line in panel B shows the percentage of TNP-8N3-ADP·Mg complex, calculated using Kd = 0.83 mM as for the ADP·Mg complex at 20 °C and pH 7.5 (53).

We wished to find out what other Na,K-ATPase ligands could affect the inactivation rate of the fluorescein enzyme and be used to evaluate the specificity of TNP-8N₃-ADP binding. Fig. 3 shows the results obtained with vanadate and strophanthidin. The latter was used (35), instead of ouabain, because facile glycoside release was needed after TNP-8N₃-ADP photoinactivation and before assaying the pNPPase activity. Likewise, to aid vanadate release, four washes were given in 150 mM NaCl, 2 mM EDTA, 20 mM Tris-Cl (pH 7.5) (36) before final resuspension in 20 mM Tris-Cl (pH 7.5), 1 mM EDTA. From Fig. 3, it is clear that when strophanthidin or vanadate binds to FITC-modified Na,K-ATPase, the TNP-8N₃-ADP photoinactivation process is slowed down considerably.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   The effects of vanadate and strophanthidin on the time course of TNP-8N3-ADP photoinactivation of FITC-modified Na,K-ATPase. FITC-modified Na,K-ATPase was photoinactivated in the presence of 20 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 25 µM TNP-8N3-ADP, with or without 200 µM orthovanadate in one experiment (open and closed circles, respectively) and with or without 200 µM strophanthidin in a separate experiment (open and closed triangles, respectively). All enzyme samples were irradiated for 30 min, with TNP-8N3-ADP added at intervals to give the indicated inactivation times. All enzyme samples were spun down in the benchtop ultracentrifuge and washed four times (a high sodium, magnesium-free wash buffer was used to accelerate vanadate release). The eight enzyme samples in each experiment were resuspended in 20 mM Tris-Cl (pH 7.5), 1 mM EDTA, and their pNPPase activity and protein concentrations were determined in triplicate. Vertical bars indicate S.E.

The effects of cations and inhibitors on the photolabeling of the fluorescein enzyme with TNP-8N₃-[-³²P]ADP were put to the test in the experiments shown in Fig. 4. As expected for the higher probe concentration, the label is distributed more widely than observed in the autoradiogram of the native enzyme (Fig. 1), as there is substantial radioactivity associated with the gel front (presumably on lipids and proteolipids) and some in relation to material that fails to penetrate the separating gel. Nevertheless, when it comes to the sodium pump subunits, it is clear that the label goes overwhelmingly to the  chain. Fig. 4, left panel, shows the cation effects. The highest labeling was obtained in the presence of 10 mM Mg²⁺ (lanes 2 and 3), followed by 150 mM Na⁺ (lane 1), which corresponds to the ranking order for inactivation of the fluorescein enzyme at the same concentration of nonradioactive TNP-8N₃-ADP (Fig. 2, A and B). Potassium ions promote a low level of photolabeling (Fig. 4, lane 5) with respect to the buffer control (lane 6) and strongly counteract the effect of Mg ions (lanes 2 and 4). Fig. 4, right panel, shows that the TNP-8N₃-[-³²P]ADP photolabeling promoted by 10 mM Mg²⁺ (lane 1) can be competed by 1 mM TNP-ADP (lane 2) and that it must, therefore, be preceded by reversible probe binding. Lanes 3 and 4 show that vanadate or strophanthidin treatment is effective at reducing the photolabeling of the fluorescein enzyme  subunit, just as it is at preventing photoinactivation of its K⁺-phosphatase activity (Fig. 3). The slight labeling of the  subunit persists in the fluorescein enzyme treated with vanadate or strophanthidin (Fig. 4, compare lanes 3 and 4 to lane 1), and this probably means that it is nonspecific. This is probably also true of the residual  chain labeling in lanes 3 and 4. Taken together with those in Fig. 3, these experiments demonstrate that two pump inhibitors, which bind from opposite sides of the membrane plane, interfere with the low-affinity incorporation of TNP-8N₃-ADP in the FITC-modified enzyme. Evidently, this second nucleotide binding locus on the  subunit is sensitive to conformational changes promoted by essential cations or key inhibitors.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Effects of pump ligands on TNP-8N3-[-32P]ADP photolabeling of the FITC-treated Na,K-ATPase. FITC-treated enzyme was photolabeled under the various conditions described below, spun down, and washed, and protein was determined in each of the final suspensions. Equal amounts (9 µg) were loaded in the gels. Left panel, fluorescein enzyme photolabeled for 30 min with 25 µM TNP-8N3-[-32P]ADP in the presence of 20 mM Tris (pH 7.5), 1 mM EDTA, and 150 mM NaCl (lane 1), 10 mM MgCl2 (lane 2), 150 mM NaCl + 10 mM MgCl2 (lane 3), 150 mM KCl + 10 mM MgCl2 (lane 4), 150 mM KCl (lane 5), and no additions (lane 6). Right panel, fluorescein enzyme photolabeled for 30 min with 50 µM TNP-8N3-[-32P]ADP in the presence of 20 mM Tris-Cl (pH 7.5), 10 mM MgCl2, and no additions (lane 1), 1 mM TNP-ADP (lane 2), 200 µM orthovanadate (lane 3), or 200 µM strophanthidin (lane 4). The arrows indicate the position of  and  chains and of the gel front (F).

The stoichiometry of TNP-8N₃-[-³²P]ADP photolabeling of the fluorescein enzyme was measured in the experiments shown in Fig. 5. The top panel shows the time course of inactivation of the K⁺-phosphatase activity of FITC-modified sodium pump caused by 25 µM TNP-8N₃-[-³²P]ADP in the presence of 100 mM NaCl; also shown is the mass of radioactive probe incorporated in  subunit bands cut from SDS-PAGE gels of the same enzyme. The direct-counting method (Fig. 5, open triangles) rested on the assumption that the mass of electrophoresed protein only consisted of  and  polypeptides, whereas the elution method (open circles) was based on the expectation that none of the TNP-8N₃-[-³²P]ADP in the excised  chain band would be released and lost through the dialysis membrane during electroelution. The first assumption was probably not too far off the mark (cf. top panels of Figs. 1 and 6); with the second method, the low total radioactivity in each excised band precluded a check for losses into the large volume of anodal solution in the electroelution tank. The expected biases should make the estimate obtained by direct counting exceed that with the elution method, and this is in part realized in Fig. 5, top panel, and in the right-hand regression in the lower panel. However, given these uncertainties, the agreement was much better than expected. The lower panel of Fig. 5 shows the long extrapolation required to assess the photolabeling stoichiometry. The results for the inactivation in Na⁺ medium (with the two incorporation estimates) were re-plotted to obtain the left-hand regression; on the right are two experiments where the inactivation was done in a medium containing 100 mM Na⁺ plus 10 mM Mg²⁺. The binding projections at full inactivation of the pNPPase activity of the fluorescein enzyme were 4.0 and 10.4 nmol of TNP-8N₃-[-³²P]ADP per mg of  chain protein, for the Na⁺ and Na⁺ plus Mg²⁺ conditions, respectively; the estimates correspond to 0.5 and 1.2 mol of TNP-8N₃-[-³²P]ADP per mol of FITC-modified  chain.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   The stoichiometry of fluorescein enzyme photolabeling. Top panel, time courses in Na+ medium. Aliquots of FITC-treated enzyme were photolabeled for periods of up to 30 min in the presence of 150 mM NaCl, 20 mM Tris-Cl, 1 mM EDTA, and 25 µM TNP-8N3-[-32P]ADP. The enzyme samples were spun down and washed; protein and pNPPase activity were determined (n = 3), and duplicate SDS-PAGE gels were run, with identical loads per channel (10 µg). Filled circles, photoinactivation of the pNPPase activity; open triangles, photolabeling of the cut  subunit band, determined by direct counting; open circles, photolabeling of the cut  subunit, determined after electroelution and protein assay. Lower panel, the correlation between photolabeling and inactivation. The photolabeling data (NaCl medium) from the top panel is replotted on the left using the same symbols as above. The right curve shows two experiments to photolabel in the presence of 100 mM NaCl plus 10 mM MgCl2, one analyzed by direct counting (filled triangles) and the other by electroelution (filled circles). The lines represent least-squares linear regressions and their 95% confidence limits. Inset, autoradiogram of an SDS-PAGE gel corresponding to a repeat of the experiment in the top panel (10 µg of protein per lane). Standard errors are shown as bars or comprised within symbols.

View larger version (72K): [in this window] [in a new window]   Fig. 6.   Partial trypsinolysis of fluorescein enzyme after TNP-8N3-[-32P]ADP photolabeling. The double-labeled Na,K-ATPase (90 µg) was incubated with 2 µg of trypsin at 37 °C in a total volume of 100 µl. After the times shown in the top panel, 10-µl samples were drawn into tubes containing 2 µl (0.5 µg) of soybean trypsin inhibitor. These samples were analyzed by SDS-PAGE. Top panel, Coomassie Blue stain; far left and far right lanes, molecular mass markers. Bottom panel, autoradiogram. The arrows on the left indicate the position of molecular mass markers (mass in kDa), of  and  chains, and of the gel front (F). The numbers on the right show the molecular mass calculated (in kDa) for the major tryptic peptides.

The result of controlled trypsinolysis of fluorescein enzyme after labeling with TNP-8N₃-[-³²P]ADP is presented in Fig. 6. The top panel shows that in the presence of Tris⁺-Cl, the  subunit quickly disappears, to generate tryptic fragments migrating very nearly as 58- and 42-kDa peptides (37), obtained with the FITC enzyme in Na⁺ or K⁺ medium (26). We also find a small amount of the 77-kDa peptide, arising in the presence of Na⁺ with the native enzyme (37), and an 81-kDa peptide. The autoradiogram in the lower panel of Fig. 6 demonstrates that the label progresses from the  chain to the 58-kDa fragment, with just a trace in the 77-kDa peptide. We conclude, therefore, that TNP-8N₃-[-³²P]ADP is incorporated in the C-terminal fragment of the  chain, downstream of Arg⁴³⁸ (38).

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

TNP-8N₃-ADP Photolabeling of the Catalytic Site-- The problem associated with low affinity binding calls for a ligand with an intrinsically higher avidity for the site in question (e.g. Ref. 39). TNP nucleotides have been extensively used not only because of their fluorescent properties (15, 40-43) but also on account of their higher binding affinity toward ATP-processing enzymes (15, 44). In the case of the sarcoplasmic reticulum Ca-ATPase, TNP-8N₃-[-³²P]ATP binds at the catalytic ATP site (20) and photolabels Lys⁴⁹² (45), equivalent to Lys⁴⁸⁰ of Na,K-ATPase (1). With the native sodium pump, we have shown that TNP-8N₃-ADP can inactivate its Na,K-ATPase activity, and the concentration required for half-maximal inactivation rate seems to be 0.05 µM (16). The results shown in Fig. 1 demonstrate that TNP-8N₃-ADP photoinactivation of the native enzyme is associated with covalent binding to the  subunit and that ATP and TNP-ADP effectively protect the site. The absence of a Na⁺ effect coincides with observations (27, 37) that the conformation of the native enzyme in Tris⁺ is close to that in Na⁺; on the other hand, the K⁺ effect probably reflects a decrease in catalytic site affinity toward TNP-8N₃-[-³²P]ADP (24, 25). At a concentration that is saturating for high affinity TNP-ADP binding (15), no TNP-8N₃-[-³²P]ADP labeling of the FITC-modified Na,K-ATPase was observed. FITC modifies the  subunit at Lys⁵⁰¹ preferentially (46), whereas 2N₃-ATP is tethered at Gly⁵⁰² (47), and 8N₃-ATP anchors at Lys⁴⁸⁰ (48). Therefore, it has seemed plausible that the sequence Lys⁴⁸⁰-Gly⁵⁰² forms part of, or is in the immediate neighborhood of, the adenine-binding region of the catalytic or high-affinity ATP site. In fact, Lys⁴⁸⁰ and Lys⁵⁰¹ can be cross-linked by H₂-DIDS, and ATP prevents this reaction (49). Other FITC anchoring residues in Na,K-ATPase are Lys⁴⁸⁰ and Lys⁷⁶⁶ (50), and there is no fundamental reason to exclude the possibility that the latter residue may also be found near the catalytic pocket. The FITC modification prevents high affinity binding of ATP (16) and formycin-triphosphate (26), a fluorescent ATP analogue. The block caused by the FITC modification, and the protection provided by ATP and TNP-ADP at equilibrium, make it quite likely that TNP-8N₃-[-³²P]ADP is labeling the catalytic site under the conditions of Fig. 1. Therefore, TNP-8N₃-[-³²P]ADP seems a suitable probe to photolabel a low affinity nucleotide site with a reasonable degree of specificity.

The FITC Block-- High and low-affinity ATP effects can be demonstrated in the soluble protomeric enzyme, in the absence of dimers or higher oligomers (6). Our approach to exploring the structural basis of a possible low affinity, or regulatory, nucleotide site has been to use the FITC modification to mask the catalytic ATP site (14, 16). The FITC block does not affect E2 partial reactions such as K⁺-phosphatase activity and P_i phosphorylation (16, 26, 28, 29), and these can still be inhibited by nucleotides with low affinity (28, 29). We decided to exploit this effect to monitor nucleotide binding at the regulatory site. The strategy called for a dependable protection of the catalytic ATP site, and we first made sure that FITC modified every  subunit in the membrane enzyme (14). Besides, the Na,K-ATPase activity, which needs ATP at both catalytic and regulatory sites (11, 12, 33), could not be rescued in the slightest degree by increasing the ATP concentration in assays with the fluorescein enzyme (14, 29). This strongly suggests that the FITC modification causes an absolute and irreversible block of the catalytic site and not just a decrease in ATP affinity. On the other hand, the apparent affinities of E2-type nucleotide effects, and toward K⁺-phosphatase substrates, genuinely decrease as a consequence of occupation of the catalytic site by FITC (14, 26, 28) or by Cr(H₂O)₄--methylene-ATP (51); in other words, full effects can be restored by increasing ligand concentrations. Further discussion on the FITC block and the hypothetical rotation of the tethered fluorescein has been published (16).

Conditions for TNP-8N₃-[-³²P]ADP Photolabeling of the Fluorescein Enzyme-- The K⁺ phosphatase activity and P_i phosphorylation of the FITC-modified enzyme can be inhibited by TNP nucleotides (14, 29), inactivated by Co(NH₃)₄ATP (51) and Co(NH₃)₄-(2')3'-O-(N-methyl-anthraniloyl)-ATP (52), and photoinactivated by TNP-8N₃-ADP (16). As both TNP-ADP (14) and TNP-8N₃-ADP (16) were found to be effective with the solubilized, FITC-modified protomer, it was proposed that the FITC-blocked site and the TNP-nucleotide binding site coexisted in the minimal enzyme unit (14, 16).

The effects of Na⁺, K⁺, and Mg²⁺ on TNP-8N₃-ADP inactivation, and labeling, of the FITC-modified enzyme were as unexpected as they were rewarding. First, there is the question of dominance: in our experiments, potassium ions have little effect in isolation, but they counteract the Na⁺ and Mg²⁺ effects (Fig. 4, left panel; see also Fig. 5 of Ref. 16). It is well known (26, 27) that the bound fluorescein can detect an Na⁺ conformation and a K⁺ conformation of the enzyme through changes in its fluorescence level. But the roles are reversed in that case, as in the presence of Tris⁺ the fluorescence of the FITC enzyme is effectively quenched by K⁺, and this is counteracted by Na⁺; on its own, Na⁺ has a small effect (26, 27). The apparent affinities are also quite different: K_0.5(Na) obtained by FITC fluorescence titrations ranged from 1.3 mM (26) to around 10 mM (27), as opposed to 97 mM in our case (Fig. 2A), whereas the K_0.5(K) estimates are 0.23 mM (26) or 0.07 mM (27), as opposed to 2.5 mM (Fig. 2C). Considering that the fluorescein enzyme could not be cycling in any of these experiments, the differences in Na⁺ affinities are more real than apparent and suggest that our observations involve an extracellular Na⁺ site. This Na⁺ site must be occupied, in addition to the high affinity Na⁺ sites involved in the fluorescence shifts, for TNP-8N₃-ADP to bind covalently to the fluorescein enzyme, i.e. the Na⁺ site regulating TNP-8N₃-ADP binding to the FITC enzyme is different from the Na⁺ site affecting the environment of the FITC molecule. Therefore, the process of TNP-8N₃-ADP photoinactivation samples an E1 fluorescein enzyme conformer that is not normally revealed by other means, one in which both "internal" and "external" Na⁺ sites are occupied. Of course, it is not possible to tell whether the high-affinity Na⁺ sites need to be filled, nor is it easy to suggest assignments for the K⁺ site (Fig. 2C). However, two situations seem clear in relation to TNP-8N₃-[-³²P]ADP incorporation in the fluorescein enzyme: (i) that it is decreased in the E2 conformation (i.e. in the presence of K⁺), and (ii) that in the absence of Mg ions, the occupation of a low-affinity Na⁺ site is an absolute requirement. These constraints reinforce the notion that that we are now dealing with a different nucleotide-binding pocket in the  chain.

With regard to the MgCl₂ effect, the apparent K_0.5(Mg) estimate of 1.3 mM (Fig. 2B) coincided with the value of 1.2 mM for the fluorescence shifts (unphosphorylated fluorescein enzyme; Ref. 27). However, this may be fortuitous, as Mg²⁺ complexation by the probe may be what matters in the present case. The increase in the level of TNP-8N₃-ADP·Mg complex in Fig. 2B, calculated by assuming a dissociation constant identical to that for ADP·Mg in these conditions (53), accounts reasonably well for the increase in inactivation rate. The imperfect fit could be due to a somewhat higher dissociation constant of TNP-8N₃-ADP·Mg (54). In Escherichia coli F₁-ATPase, TNP-ATP·Mg binds to all three catalytic sites with higher affinity than TNP-ATP (44); only the Mg complex binds at noncatalytic sites (55).

The effects of vanadate and strophanthidin in protecting the FITC-modified enzyme from TNP-8N₃-ADP labeling (Fig. 4) and inactivation (Fig. 3) suggest that these inhibitors stabilize the pump in nonreactive conformations that are equivalent, although not necessarily the same. In the presence of Mg²⁺ but in the absence of Na⁺, which inhibits vanadate binding (56), vanadate stabilizes the enzyme in an E2 conformation both before (36, 57) and after (58) FITC treatment. The same situation arises with ouabain (26, 27, 59); in this case, the transition occurs slowly (cf. Fig. 4) but is not easily reversed (26, 27). These are indeed not the usual E2 form representing a stage in the overall Na,K-ATPase reaction. Ouabain binding generates a very stable E2 conformation (59), and vanadate presumably behaves as a transition-state analogue of phosphoenzyme hydrolysis (57). In the process, vanadate competes with ATP at a low-affinity site (57). Mutual exclusion between phosphoenzyme and low-affinity nucleotide binding may also explain the inactivation of P_i phosphorylation by TNP-8N₃-ADP in the fluorescein enzyme (16).

Specificity of Fluorescein Enzyme Photolabeling-- The stoichiometry of TNP-8N₃-[-³²P]ADP labeling had an upper limit close to 1 mol of probe per mol of  chain. The higher value in Mg²⁺ medium may in part reflect a higher initial labeling. However, it seems that TNP and azido nucleotides are slowly lost from the enzyme (45, 48, 60), and it is conceivable that the incorporation of TNP-8N₃-[-³²P]ADP·Mg simply be more stable than that of the free probe. In addition, the observed stoichiometry in the presence of Mg²⁺ may be a balance between specific and nonspecific labeling, on the one hand, and label loss, on the other. In fact, when the total radioactivity incorporated in the purified, membrane-bound preparation was considered, the labeling was roughly doubled. This is evident from the radioactivity associated with the gel front (Figs. 4-6), which is greater when Mg²⁺ is included in the inactivating solution. Of necessity, vanadate and strophanthidin protection experiments (Fig. 4B) were done in the presence of Mg²⁺, and this in itself may have increased the nonspecific binding. The actual results obtained with vanadate and strophanthidin, however, indicate that about half of the  chain labeling can be prevented by the inhibitors and suggest that at least this much represents specific incorporation. Nonspecific labeling in the presence of Na⁺ alone is likely to be a smaller fraction of the total binding observed (Fig. 5).

Conclusions-- Low concentrations of TNP-8N₃-[-³²P]ADP photolabel the  chain of native Na,K-ATPase, most probably from within the high affinity site. On the other hand, the inactivating effects of higher TNP-8N₃-ADP concentrations on the FITC-modified enzyme had been found to be consistent with probe binding at a low affinity nucleotide site (16). The experiments that we now report demonstrate that the incorporation of the radioactive TNP-8N₃-ADP in the fluorescein enzyme complies exactly with the behavior expected from that inactivation study. The labeling is essentially confined to the  subunit, and a large proportion appears to be specific. This approach, therefore, seems appropriate to try and map the location of a regulatory ATP site on the  chain structure. Partial trypsinolysis has shown that the TNP-8N₃-[-³²P]ADP label is located in the C-terminal two-thirds of the FITC-modified  subunit, which also contain other sites normally involved in the enzymatic processes of the pump. We are now purifying the ³²P-labeled peptides resulting from extensive proteolysis of the FITC-modified  chain, to obtain the sequence of the TNP-8N₃-[-³²P]ADP anchoring fragment(s).

	ACKNOWLEDGEMENTS

We thank Prof. Paul Cullis (Dept. of Chemistry, University of Leicester) for his advice on the 8N₃-ATP synthesis, Dr. Blair Grubb for the computer handling of the gel figures, and Tim Walton for his help with the enzyme purification.

	FOOTNOTES

^* This work was supported by a research grant from The Wellcome Trust.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.

To whom correspondence should be addressed. Tel. and Fax: 44-116-2523091; E-mail: jdc7{at}le.ac.uk.

The abbreviations used are: Na, K-ATPase, sodium- and potassium-dependent adenosine triphosphatase (EC 3.6.1.37); FITC, fluorescein 5'-isothiocyanate; pNPP, p-nitrophenyl phosphate; pNPPase, p-nitrophenyl phosphatase; TNP-ADP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate; TNP-8N₃-ADP, 2'(3')-O-(2,4,6-trinitropheyl)8-azidoadenosine 5'-diphosphate; E1, Na⁺ form of enzyme; E2, K⁺ form of enzyme.

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