©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Coupling of Phosphorylation and Nucleotide Binding Sites in the Proteolytic Fragments of Na/K-ATPase (*)

(Received for publication, August 25, 1994; and in revised form, November 21, 1994)

Nina Zolotarjova Sankaridrug M. Periyasamy Wu-Hsiung Huang Amir Askari

From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cleavage of the alpha-subunit of Na/K-ATPase by trypsin at Arg-Ala causes enzyme inhibition which has been suggested to be due to altered alignment of phosphorylation site on the 48-kDa N-terminal fragment with nucleotide binding site on the 64-kDa C-terminal fragment. Our aims were to test this hypothesis and to assess the effect of the cleavage on the enzyme's two ATP sites. Na-dependent phosphorylation of the partially cleaved enzyme by ATP showed that K(0.5) values of ATP for phosphorylations of intact alpha and 48-kDa peptide were the same (0.4 µM). Unchanged interactions among the residues across the cleavage site were also indicated by data showing that reaction of fluorescein isothiocyanate with the 64-kDa peptide blocked phosphorylation of the 48-kDa peptide by ATP.

ATP is known to block the reaction of fluorescein isothiocyanate with the enzyme. Experiments on the partially cleaved enzyme showed that K(0.5) of ATP for protection of alpha was 30-60 µM, and the value for the protection of interacting 48-kDa and 64-kDa peptides was 1-3 mM. Evidently, while the cleavage does not affect the high affinity catalytic site, it disrupts the allosteric low affinity ATP site.

Experiments on reconstituted preparations showed that the cleavage abolished ATP-dependent Na/K exchange, P(i) + ATP-dependent Rb/Rb exchange, ATP-dependent Na/Na exchange, and ADP + ATP-dependent Na/Na exchange activities. Selective disruption of the low affinity ATP site accounts for the inhibitions of all functions involving K(Rb), based on the established role of this site in the control of K access channels. Cleavage-induced inhibitions of other activities, however, suggest additional roles of the low affinity ATP site in the reaction cycle.


INTRODUCTION

Controlled proteolysis of the alpha-subunit of Na/K-ATPase has provided invaluable information about the structural organization of the enzyme within the plasma membrane(1, 2) . Studies on the functional changes induced by controlled proteolysis have also been of help in generating hypotheses on the enzyme's structure-function relationships(3, 4, 5, 6) . Of particular interest is the specific cleavage between Arg and Ala when the purified kidney enzyme is exposed to trypsin in the presence of K. Because this cleavage site is between several residues (in the region of amino acids 470-720) that are involved in ATP binding and Asp which is phosphorylated and dephosphorylated in the course of ATP hydrolysis(2, 7) , it is reasonable to think that the cleavage may affect the functional coupling between the ATP binding site and the phosphorylation site. In fact, based on a number of studies(3, 4, 8) , it has been proposed that this cleavage interferes with the orientation of the two sites and reduces affinity for nucleotide binding at the catalytic site, while it allows the operation of several transport functions of the enzyme that require ATP binding and phosphorylation (3, 4) . We were prompted to re-examine the functional consequences of this cleavage because previous work had not addressed the relation of the cleavage to the multiple nucleotide binding sites of the enzyme. That the native enzyme exhibits two nucleotide binding sites with widely different affinities has been known for a long time(9, 10, 11) . Uncertainties remain, however, on whether the two different affinities are those of the different conformational states that appear in sequence during the reaction cycle or are due to the simultaneous existence of two distinct binding sites on the functional unit of the enzyme(11) . The suggested effect of the trypsin cleavage on ATP affinity seemed to provide an opportunity for clarifying the relationships between the two nucleotide sites and the phosphorylation site.


EXPERIMENTAL PROCEDURES

Purified membrane-bound Na/K-ATPase of canine kidney medulla, with specific activity in the range of 800-1600 µmol of ATP hydrolyzed/mg of protein/h, was prepared as described before(12) . The enzyme (0.5 mg/ml) was exposed to trypsin at 37 °C in a solution containing 10 mM KCl, 15 mM Tris-HCl (pH 7.4), and 0.3-0.5 µg of trypsin. Unless indicated otherwise, reaction time was chosen to achieve about 50% inactivation of the enzyme. The reaction was stopped by the addition of trypsin inhibitor (0.7 mg/µg of trypsin). The enzyme was collected by centrifugation in cold, washed with 10 mM Tris-HCl (pH 7.4), and suspended in 0.25 M sucrose, 1 mM EDTA, and 30 mM histidine (pH 6.8) prior to use.

Reaction with FITC (^1)was carried out by incubating the native or the partially cleaved enzyme (1 mg/ml) at 20 °C for 30 min in a solution containing 100 mM Tris-HCl (pH 9.2), 1 mM EDTA, and the indicated concentrations of FITC. The reaction was terminated by 50-fold dilution with cold 50 mM Tris-HCl (pH 7.2) and centrifugation at 100,000 times g. After an additional wash in the same buffer, the enzyme was suspended in 0.25 M sucrose, 1 mM EDTA, and 30 mM histidine (pH 6.8) and either used or stored frozen for subsequent use. Under these incubation conditions, 45 µM FITC caused more than 95% inhibition of Na + K-dependent ATPase activity. For the detection of FITC-labeled peptides, the enzyme samples were subjected to SDS-gel electrophoresis under alkaline conditions(13) , and the unstained gels were illuminated with UV light and photographed.

ATPase assays were done at 37 °C through the determination of the initial rate of release of P(i) from [-P]ATP(12) . Reaction mixture for Na + K-dependent ATPase contained 2 mM ATP, 3 mM Mg, 100 mM Na, 20 mM K, 1 mM EGTA, and 50 mM Tris-HCl (pH 7.4). For Na-dependent ATPase, K was omitted, and 0.1 mM ATP was used. Na-dependent ADP/ATP exchange was assayed at 37 °C by measuring the rate of incorporation of [^14C]ADP into ATP as described before(14) . Enzyme phosphorylation by [-P]ATP was done at 0 °C as described before (12) , by incubating the enzyme in the presence of 2 mM Mg, 100 mM Na, and the indicated ATP concentration for 90 s. This was sufficient to achieve the maximal level of phosphoenzyme. Phosphorylation by P(i) was done at 37 °C for 10 min in the presence of 2 mM Mg and 50 mM Tris-HCl (pH 7.2) and the indicated P(i) concentration, as described(12) . The acid-denatured phosphorylated enzymes were either filtered and counted (12) or subjected to SDS-gel electrophoresis at pH 2.4(5) . The gels were stained and autoradiographed by conventional procedures. The bands were quantified by a soft laser scanning densitometer. Multiple exposures of the radioactive bands were analyzed to ensure that the signals were within the linear range of the film. ADP binding to the enzyme was measured at 0 °C by the centrifugation method(15) , in a medium containing 50 mM Tris-HCl (pH 7.4), 5 mM NaCl, 0.1 mM EDTA, and the indicated concentrations of [^14C]ADP. The enzyme solubilized with CE(8) was prepared according to Esmann (16) and phosphorylated as the native enzyme but in reaction mixtures containing 0.15% CE(8).

For the assay of transport activities, the enzyme was solubilized with CHAPS and reconstituted by the following modifications of previous procedures(17) . The enzyme was washed in buffer A containing 160 mM KCl and 20 mM MOPS (pH 7.2), and suspended (1 mg/ml) in the same buffer containing 1% CHAPS and 20% glycerol. After constant stirring on ice for 40 min, the suspension was centrifuged at 130,000 times g for 30 min. The soluble supernatant was mixed with soybean phospholipids (20 mg/ml) and vortexed to obtain a uniform suspension. The suspension (0.5 ml) was placed on a 5-ml Sephadex G-25-300 column which had been equilibrated with buffer A, allowed to remain at 4 °C for 60 min, and centrifuged at 1,500 times g for 10 min. The collected proteoliposomes from several columns were pooled and centrifuged at 150,000 times g for 60 min. The sedimented proteoliposomes were mixed with buffer A to obtain a suspension of 1-2 µg of protein/µl. To assay ATP-dependent Na/K exchange, 20 µl of this suspension was added to 180 µl of a reaction mixture containing 150 mM choline chloride, 5 mMNaCl, 0.2 mM EGTA, 10 µM valinomycin, 3 mM MgCl(2), 2 mM ATP, 20 mM MOPS (pH 7.2). ATP was omitted from the control. After 20 s of incubation at 23 °C, the reaction was stopped by adding 3 ml of ice-cold buffer A, and the mixture was passed through a 0.45-µm Millipore filter. The filter was washed twice with 3 ml of cold buffer A and counted. ATP-dependent Na uptake was calculated assuming linearity within the first 20 s. It was established that, under the conditions used here, the time course of ATP-dependent Na uptake by these proteoliposomes was similar to that described before(31) . Proteoliposomes to be used for Na/Na exchange were prepared as above, except that buffer A was replaced with one containing 200 mM NaCl and 10 mM Tris-HCl (pH 7.2). For the assay of ATP-dependent Na/Na exchange, the reaction mixture contained 5 mMNaCl, 3 mM MgCl(2), 2 mM ATP, and 205 mM Tris-HCl (pH 7.2). For the assay of ATP + ADP-dependent exchange, the reaction mixture was the same, except that it contained 1 mM ATP + 1 mM ADP. Appropriate controls without ATP and ADP were included. Incubation time was 2 min. For the assay of Rb/Rb exchange, proteoliposomes were prepared similarly, except that buffer A was replaced with one containing 25 mM RbCl(2) and 100 mM Tris-HCl (pH 7.2). The complete reaction mixture contained 10 mMRbCl, 6 mM MgCl(2), 5 mM P(i), 1 mM ATP, and 100 mM Tris-HCl (pH 7.2). Reaction time was 1 min. Controls without P(i) and ATP were included, and it was established that both P(i) and ATP were required for the stimulation of uptake. Protein in proteoliposomes was measured according to Kaplan and Pedersen(18) .

Trypsin (type III-S, bovine pancreas), trypsin inhibitor (soybean), ATP, FITC, and crude soybean phospholipid IV were obtained from Sigma. All radioisotopes were purchased from DuPont NEN. P(i) was purified before use(12) .


RESULTS

Incubation of the enzyme with trypsin and K resulted in the cleavage of the alpha-subunit into two fragments with molecular masses of about 64 kDa and 48 kDa (Fig. 1a). When the incubation time with trypsin was varied in a number of experiments, and the disappearance of intact alpha was correlated with Na + K-dependent ATPase activity (Fig. 2), it became apparent that the cleavage caused complete inhibition of this activity.


Figure 1: Cleavage of Na/K-ATPase by trypsin in the presence of K and Na-dependent phosphorylation of the cleaved enzyme by ATP. Control enzyme (lanes 1 and 2) and partially cleaved enzyme (lanes 3 and 4) were exposed to 1 µM [-P]ATP in the presence of either Na (lanes 1 and 3) or K (lanes 2 and 4). Acid-denatured samples were subjected to SDS-gel electrophoresis at pH 2.4. The gels were stained (a) and autoradiographed (b).




Figure 2: Relation between activity and remaining alpha after exposure to trypsin and K. Enzyme samples were exposed to trypsin for different periods. Remaining alpha was estimated by densitometry of stained gels, and Na + K-dependent ATPase was assayed under standard conditions.



On the 7.5% acid gels of Fig. 1a, the 64-kDa peptide was not clearly separated from the intact beta-subunit. Such separation was achieved on 5% gels, allowing the determination of the N-terminal sequence of the 64-kDa peptide. This turned out to be XVAGDA, consistent with the cleavage occurring between Arg and Ala of the canine kidney alpha sequence(19) . This location is in agreement with that of the cleavage point in the pig kidney enzyme(20) .

Phosphorylations of Intact and Cleaved alpha-Subunits by ATP

In experiments of Fig. 1b, the partially cleaved preparation was exposed to [-P]ATP in the presence of Mg + Na or Mg + K, subjected to electrophoresis on acid gels, and autoradiographed. The results confirmed previous findings(3) , showing that the 48-kDa peptide, like the intact alpha, is phosphorylated by ATP in a Na-dependent manner.

Na-dependent phosphorylation of the native enzyme by ATP involves nucleotide binding to the high affinity catalytic site. Based on indirect evidence, the affinity of this site was postulated to be reduced after the trypsin cleavage(3, 8) . To test this hypothesis directly, the partially cleaved enzyme was phosphorylated in the presence of different concentrations of [-P]ATP, and the extent of P-labeling of the intact alpha and the 48-kDa peptide was determined on acid gels. The inset to Fig. 3shows the gels obtained in one such experiment. The combined data of similar experiments with seven different partially cleaved preparations (Fig. 3) showed that K(0.5) value of ATP for phosphorylation of the 48-kDa peptide was not different from that of the intact alpha, indicating that the high affinity ATP binding site is not disturbed by the cleavage. (^2)


Figure 3: Effects of varying ATP concentrations on the phosphorylations of intact alpha and 48-kDa fragment. Inset, a partially cleaved enzyme was phosphorylated in the presence of Na by [-P]ATP under standard conditions. ATP concentrations (µM) were (lanes 1-6, respectively): 0.1, 0.2, 0.5, 1, 3, and 5. Equal protein samples were resolved on acid gels. Gels were stained (a) and autoradiographed (b). Graph, combined data of 7 different experiments similar to the inset. Phosphopeptide levels were measured by densitometry of autoradiograms. Values are mean ± S.E., expressed as percent of the maximal level at 5 µM ATP.



These results, in conjunction with those of Fig. 2, implied that the cleaved alpha-subunits that bind ATP with high affinity and are phosphorylated do not participate in Na + K-dependent ATPase activity. To test this directly, in a number of partially cleaved preparations, Na + K-dependent ATPase activity and the maximal level of phosphoenzyme formation from ATP were determined and compared with the corresponding control values. In all partially cleaved preparations, the remaining phosphoenzyme exceeded the remaining ATPase activity (Fig. 4). That the total phosphoenzyme was reduced at all in the cleaved preparations is most likely due to the secondary cleavage of the 48-kDa peptide(8) . In preparations that had lost ATPase activity by more than 70%, we consistently noted additional peptide bands with mobilities higher than those of the 48-kDa peptide (not shown).


Figure 4: Relation between Na + K-dependent ATPase activity, total acid-stable phosphoenzyme, and maximal capacity for ADP binding after trypsin cleavage. Enzyme preparations exposed to trypsin plus K were assayed under conditions described in the text.



To compare the responses of the intact and the cleaved alpha to Na and K, experiments of Fig. 5were done. The partially cleaved enzyme was phosphorylated with saturating ATP in the presence of different Na concentrations, and the phosphorylated bands were quantitated on acid gels. The Na activation curves for the intact alpha and the 48-kDa peptide were superimposable (Fig. 5a). In similar experiments, the inhibitory effects of varying K concentrations on Na-dependent phosphorylations of the intact alpha and the 48-kDa peptide were also found to be nearly identical (Fig. 5b).


Figure 5: Effects of varying Na and K concentrations on phosphorylations by ATP of intact alpha and 48-kDa fragment. a, experiments were done as in Fig. 3, in the presence of 5 µM ATP and the indicated Na concentrations. Values are means and ranges of 3 experiments, expressed as percent of the highest value obtained at 50 mM Na. b, experiments were done as in a, in the presence of 50 mM Na and the indicated concentrations of K.



ADP Binding to Intact and Cleaved Enzymes

ADP also binds to the high affinity ATP site(15) . When the bindings of [^14C]ADP to the control enzyme and a partially cleaved preparation were measured at different ADP concentrations (0.2-20 µM) as indicated under ``Experimental Procedures,'' the dissociation constants evident from the resulting plots were 2.1 µM and 2.3 µM, respectively. This apparent lack of effect of the cleavage on high affinity ADP binding is in agreement with previous data (8) showing equal affinities of the control and the partially cleaved enzymes for ATP binding to the high affinity site. One possible explanation for these findings is that the cleavage abolishes high affinity nucleotide binding (or greatly reduces its affinity) so that the detected binding to the partially cleaved enzyme is solely due to the remaining uncleaved enzyme. The alternative is that the control and the cleaved enzymes have the same affinity at the high affinity site. To distinguish between the alternatives, in two partially cleaved enzymes and controls, Na + K-dependent ATPase activities and [^14C]ADP binding capacities at 20 µM ADP were compared. In both cleaved preparations, the remaining ADP binding capacities exceeded the remaining ATPase activities (Fig. 4). These data support the conclusions of the phosphorylation experiments of Fig. 3and Fig. 4, indicating that the high affinity nucleotide binding site is not affected by the cleavage.

FITC Interactions with Intact and Cleaved alpha-Subunits

FITC inhibits Na + K-dependent ATPase activity of the enzyme irreversibly by reacting with one or more lysine residues at or near the nucleotide binding sites of the alpha-subunit(2, 21, 22, 23, 24, 25) . There is evidence to indicate that FITC interacts with both high and low affinity nucleotide sites. In the FITC-labeled enzyme, the high affinity ATP site is not detected(21) , and the affinity of the low affinity site is reduced(26) . Also, reaction of FITC with the enzyme is prevented by ATP at the low affinity site(27, 28) . Proteolytic cleavage of the FITC-labeled enzyme has been studied(21, 29) . In order to learn more about the effects of the trypsin cleavage on the phosphorylation and the nucleotide binding sites, in the following experiments we cleaved the enzyme first and then studied the interactions of the partially cleaved enzyme with FITC and ATP.

A partially cleaved enzyme preparation was reacted with FITC to obtain inhibition of the remaining Na + K-dependent ATPase activity. When this preparation was subjected to gel electrophoresis, the fluorescence of the incorporated fluorescein was detected in the intact alpha and the 64-kDa C-terminal peptide, but not in the 48-kDa peptide (Fig. 6). This is consistent with the locations of the lysine residues of the intact alpha that have been shown to react with FITC(2) .


Figure 6: Reaction of FITC with intact alpha and 64-kDa fragment. A partially cleaved enzyme (lanes 1 and 2) and a control enzyme (lane 3) were reacted with FITC and subjected to electrophoresis on alkaline gels. Lane 1, stained with Coomassie blue. Lanes 2 and 3, UV-illuminated gels.



Reaction of FITC with the native enzyme blocks high affinity ATP binding and the Na-dependent phosphorylation by ATP(21, 22) . In experiments of Fig. 7, samples of the partially cleaved enzyme were exposed to different FITC concentrations for a fixed time period and washed. After phosphorylation with 1 µM [-P]ATP, the samples were resolved on acid gels and autoradiographed. The results (Fig. 7) clearly showed that reaction with FITC blocks phosphorylations of both the intact alpha and the 48-kDa peptide. Quantitation of the autoradiograms of Fig. 7indicated equal sensitivities of the phosphorylations of intact alpha and 48-kDa peptide to varying FITC concentrations. The K(0.5) value of FITC estimated from Fig. 7is about the same as the K(d) value (5.7 µM) calculated previously from inactivation rate constants(22) .


Figure 7: Inhibitory effects of varying FITC concentrations on Na-dependent phosphorylations of alpha and 48-kDa fragment by ATP. Samples of a partially cleaved preparation were reacted with different FITC concentrations for a fixed period and then exposed to [-P]ATP as described in the text. Equal amounts of protein were applied to acid gels. a, stained gel. b, autoradiograms of samples reacted with varying FITC concentrations (µM): 0, 3, 5, 10, 20 (lanes 1-5, respectively).



The combined data of Fig. 6and Fig. 7indicate that the reaction of FITC with the 64-kDa peptide blocks high affinity ATP binding to the interacting 64-kDa and 48-kDa peptides and prevents the phosphorylation of the 48-kDa peptide.

To see if detergent solubilization affects the interaction between 64-kDa and 48-kDa peptides, the following experiments were done. The partially cleaved enzyme was reacted with FITC to obtain complete inhibition of its remaining ATPase activity. This, and the partially cleaved preparation not reacted with FITC, were then solubilized with CE(8) as indicated under ``Experimental Procedures'' and exposed to [-P]ATP as in the experiments of Fig. 7. The results (not shown) were identical with those seen with the membrane-bound preparation. While the 48-kDa peptide of the control-solubilized preparation was phosphorylated, that of the solubilized FITC-labeled preparation was not phosphorylated. Evidently, interaction between the 48-kDa and 64-kDa peptides is not disturbed by solubilization.

ATP prevents the reaction of FITC with the native enzyme(21, 24) . This protection, however, requires such high concentrations of ATP that it suggests the involvement of the low affinity ATP site(27, 28) . To compare the protective effects of ATP on FITC reactions with intact alpha and 64-kDa peptide, samples of the partially cleaved enzyme were reacted for a fixed period with a fixed concentration of FITC in the absence of ATP and in the presence of varying ATP concentrations (0.01-2.5 mM). After washing, fluorescence of the fluorescein incorporated into intact alpha-subunit and 64-kDa peptide were detected as in Fig. 6and quantitated by densitometry. The results (Fig. 8) showed clearly that ATP was far more effective in preventing FITC reaction with intact alpha than with the 64-kDa peptide.


Figure 8: Prevention of FITC reaction with alpha and 64-kDa fragment by varying concentrations of ATP. Samples of partially cleaved enzyme were reacted with 45 µM FITC for 30 min in the absence of ATP and in the presence of indicated ATP concentrations. After electrophoresis at alkaline pH, fluorescent bands were detected as in Fig. 6and measured by densitometry.



Since FITC reacts with different classes of sites on the enzyme(24) , it was necessary to assess the functional relevance of the sites whose reactions with FITC were prevented by ATP in the above experiments. Therefore, another set of experiments similar to those of Fig. 8was conducted. After reaction of the partially cleaved enzymes with FITC in the presence of varying ATP concentrations, excess ATP and FITC were removed by washing, each sample was phosphorylated with 1 µM [-P]ATP, and the levels of P-labeled alpha and 48-kDa peptides were determined on acid gels (Fig. 9). The results showed clearly that (a) when unlabeled ATP was present during the reaction of FITC with the enzyme, subsequent phosphorylation with labeled ATP was greater and (b) at each ATP concentration, protection of alpha was greater than that of the 48-kDa peptide. From the data of Fig. 8and quantitation of the results of Fig. 9, the approximate K(0.5) value of ATP for the protection of alpha was 30-60 µM, and the value for the protection of 64-kDa and 48-kDa peptides was 1-3 mM. Evidently, trypsin cleavage causes a significant decrease in the affinity of a low affinity ATP site of the enzyme.


Figure 9: Protective effects of varying ATP concentrations on the FITC-induced inhibition of Na-dependent phosphorylations of alpha and 48-kDa fragment by ATP. Partially cleaved enzyme was reacted with FITC as in Fig. 8in the absence of ATP and in the presence of different ATP concentrations. After removal of excess FITC and ATP, the samples were reacted with [-P]ATP in the presence of Na, and the acid-denatured samples were resolved on acid gels and autoradiographed. Two sets of experiments were done using different ranges of ATP concentrations during reaction with FITC. Representative stained gels of the two sets are shown in a and c. Autoradiograms of the two sets are shown in b and d. In each set, lane 1 is the enzyme that was not reacted with FITC. In b, ATP concentrations (µM) during reaction with FITC were: 0, 10, 20, 50, 100 (lanes 2-6, respectively). In d, ATP concentrations (mM) during reaction with FITC were: 0, 0.1, 0.5, 1, and 5 (lanes 2-6, respectively).



Interactions of P(i) and AMPPCP with Intact and Cleaved alpha-Subunits

Asp of the native alpha-subunit is also phosphorylated by P(i) in the presence of Mg(12) , and inhibition of this phosphorylation by nucleotides involves a low affinity nucleotide site(30) . It was of interest, therefore, to compare the interactions of P(i) and nucleotides with intact and cleaved alpha-subunits.

Experiments of Fig. 10a with a partially cleaved enzyme showed that at 2 mM Mg, the K values of P were nearly the same for phosphorylations of intact alpha and 48-kDa peptide and similar to the value for phosphorylation of the native enzyme at 2 mM Mg(12) .


Figure 10: Effects of varying P(i) concentrations on phosphorylations of alpha and 48-kDa fragment (a) and inhibitory effects of AMPPCP on phosphorylations of alpha and 48-kDa fragment (b). In a, the partially cleaved enzyme was reacted with P(i) in the presence of 2 mM Mg. The acid-denatured samples were resolved on acid gels and quantitated as in Fig. 3. In b, the partially cleaved enzyme was phosphorylated with 10 µMP(i) in the absence of nucleotide and in the presence of the indicated concentrations. Mg concentration was 2 mM.



For technical reasons, it is difficult to use ATP to study the low affinity inhibitory effects of nucleotides on P(i) phosphorylation(30) . In experiments of Fig. 10b, the effects of AMPPCP on phosphorylations of the intact alpha and 48-kDa peptide by P(i) were studied in a partially cleaved preparation. The results of Fig. 10clearly suggest that the trypsin cleavage causes a significant decrease in the affinity of an AMPPCP binding site.

Transport Activities and the Associated Partial Reactions of Intact and Partially Cleaved Enzymes

Since previous data on the transport functions of the trypsin-cleaved enzyme were limited and inconclusive (Table III of (4) ), for the further assessment of the functional consequences of the altered low affinity nucleotide binding site, reconstituted preparations of intact and partially cleaved enzyme were used to assay ATP-dependent Na/K exchange, P(i) + ATP-dependent Rb/Rb exchange, ATP-dependent Na/Na exchange, and ATP + ADP-dependent Na/Na exchange. All four activities were inhibited by the cleavage, and there were no significant differences among their inhibitions (Table 1).



While the ATP-dependent Na/K exchange reflects the Na + K-dependent ATPase activity, the ATP-dependent Na/Na exchange and ADP + ATP-dependent Na/Na exchange represent the Na-dependent ATPase and Na-dependent ADP/ATP exchange activities, respectively (31) . As expected from the data of Table 1, experiments of Table 2showed no significant differences among the cleavage-induced inhibitions of the two ATPase and the ADP-ATP exchange activities. Our finding on the inhibition of ADP-ATP exchange by the trypsin cleavage is in agreement with a previous report(32) .



Cleavage-induced Inhibition of Na + K-dependent ATPase at Different ATP Concentrations

Since the above experiments indicated the disruption of a low affinity nucleotide site, but not that of the catalytic site, by trypsin cleavage, Na + K-dependent ATPase activities of partially cleaved and control preparations were compared at six different ATP concentrations in the range of 0.05-5 mM. Percent inhibitions were the same at all concentrations (data not shown).


DISCUSSION

Structural Relations of the Two ATP Sites

Our data showing that ATP phosphorylates the 48-kDa fragment with the same high affinity that it phosphorylates the intact alpha-subunit (Fig. 3) and the supporting data on high affinity ADP binding ( Fig. 4and ``Results'') clearly indicate that the ensemble of residues involved in high affinity ATP binding and enzyme phosphorylation is not disturbed by the trypsin cleavage, in spite of the fact that the components of the ensemble are located on both sides of the cleavage site. Continued interaction among the residues on the two sides of the cleavage site is also indicated by the data showing that reaction of FITC with the 64-kDa fragment prevents phosphorylation of the 48-kDa fragment by ATP ( Fig. 6and Fig. 7). That this phenomenon persists after detergent solubilization (see ``Results'') suggests the high stability of the continued interaction between the proteolytic fragments.

In sharp contrast to the high affinity ATP binding site, the low affinity ATP binding site whose occupation protects against FITC inactivation is clearly disrupted by the trypsin cleavage ( Fig. 8and 9). Since FITC inactivation experiments are done at pH 9.2, and ATP phosphorylation experiments at pH 7.2, one may suspect that pH differences account for the above contrast. That this is not the case, however, is indicated by the fact that disruption of the low affinity nucleotide site by the cleavage is also noted in P(i) phosphorylation experiments done at pH 7.2 (Fig. 10).

The most obvious explanation for the selective disruption of the low affinity site by the trypsin cleavage is that the two nucleotide sites are distinct physical entities. The phenomenon may also be explained by assuming that the two sites are on two interconvertible conformational states and that the cleavage is disruptive to one conformation but not the other. The latter alternative, however, is not consistent with previous data showing the simultaneous bindings of nucleotides to the two sites(26, 33, 34) . Therefore, if the two sites are indeed slightly different states of the same ensemble of residues(35) , it is necessary to assume that the two states are not mutually exclusive; i.e. there must be a hybrid species containing the two conformational states.

Consequences of the Disruption of the Low Affinity Site

It is convenient to consider the functional consequences of the disruption of the low affinity site in the context of the widely used Albers-Post reaction cycle. In a simple version of this cycle for Na + K-dependent ATPase and Na/K exchange (Fig. 11), the high affinity site is the catalytic site on E(1) at which ATP binds and phosphorylates the enzyme, and the low affinity site is on E(2) where ATP binds to widen the access channels for K(36) , allowing the release of occluded K from the stable E(2)(K) and the conversion of E(2) to E(1). In this scheme, Na-dependent ATP/ADP exchange and ATP + ADP-dependent Na/Na exchange are due to reactions 1 and 2; P(i) + ATP-dependent K/K exchange (or Rb/Rb exchange) is due to reactions 3 and 4; and Na-dependent ATPase and ATP-dependent Na/Na exchange are due to the entire cycle when K is replaced with Na(31) .


Figure 11: The Albers-Post cycle of Na + K-dependent ATPase activity. Na and K are intracellular ions, and Na and K are extracellular ions.



The observed inhibitions of Na+ K-dependent ATPase activity ( Fig. 2and Table 2) and ATP-dependent Na/Kexchange (Table 1) resulting from trypsin cleavage are not due to any cleavage-induced change in the affinity of ATP at the high affinity catalytic site (Fig. 3). The apparent affinity of Nafor phosphorylation of the cleaved enzyme, and its apparent Kaffinity, are also not significantly different from the corresponding Naand Kaffinities of the intact enzyme (Fig. 5), and the normal Na-induced and K-induced fluorescence responses of the cleaved enzyme have also been reported(4) . The inhibitions of the two physiologically prominent activities of the enzyme by the cleavage are consistent, however, with the selective disruption of the low affinity ATP site since this site is involved in the normal cycle of these activities (Fig. 11). That inhibition of Na+ K-dependent ATPase of the cleaved enzyme is independent of ATP concentration (see ``Results''), however, suggests that the damage done by the cleavage is not a simple matter of reduced ATP affinity at the low affinity site. It is likely that the coupling mechanism between the site and the Kaccess channels of E(2)(36) is also disturbed by the cleavage.

The inability of the cleaved enzyme to carry out P(i) + ATP-dependent Rb/Rb exchange (Table 1) is also expected since this mode of exchange requires ATP only at the low affinity site. The observed inhibitions of Na-dependent ATPase, Na-dependent ADP/ATP exchange, and the Na/Na exchange activities by the trypsin cleavage ( Table 1and Table 2), however, are more difficult to explain. Although the relation of a disrupted low affinity nucleotide site to these activities is not apparent from Fig. 11, there are previous studies that implicate the site in these partial reactions. The K(0.5) values of ADP for the activations of Na-dependent ADP/ATP exchange and ADP + ATP-dependent Na/Na exchange are significantly higher than the dissociation constant of ADP for the high affinity nucleotide site, suggesting the possibility of the participation of both high and low affinity nucleotide sites in these reactions(37, 38) . In the case of Na-dependent ATPase activity and the corresponding ATP-dependent Na/Na exchange, the hyperbolic ATP curves and the K(0.5) values (39, 40) are consistent with the involvement of only the high affinity ATP site; however, substrate inhibition noted at higher ATP concentrations in several studies(39, 41) , observations on the turnover of the phosphoenzyme at different ATP concentrations(42) , and the studies on the combined effects of P(i) and ATP on Na-dependent ATPase (43) suggest the existence of the low affinity ATP site during the reaction cycle of Na-dependent ATPase. Our data suggest, therefore, that even if the low affinity site need not be fully occupied, its disruption by trypsin cleavage prevents the completion of the Na-dependent ATPase cycle. The recent observations on the involvement of the low affinity ATP site in Na-dependent phosphorylation (44, 45) may prove to be relevant to the clarification of the role of this site in the various Na-dependent activities of the enzyme.

It is appropriate to point out that in alternatives to the Albers-Post cycle, i.e. the variations of the bicyclic scheme(46, 47) , it is also difficult to reconcile the selective disruption of the low affinity site with inhibitions of Na/Na exchange activities and Na-dependent ATPase.

Conclusions

The long established role of the low affinity ATP site in K deocclusion (Fig. 11) has attracted such attention that we may have overlooked additional roles of this site in the function of the enzyme. The most general conclusion to be drawn from our data is that the coupling of the low affinity site to the high affinity and phosphorylation sites seems to be essential for most catalytic functions of the enzyme. Whether the pathways of this and related coupling mechanisms are within a single alpha-chain, or across the boundaries of alpha^n, or both(36) , remains to be established.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL-36573 awarded by NHLBI, United States Public Health Service, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: FITC, fluorescein isothiocyanate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; AMPPCP, adenosine 5`-(beta,-methylene)triphosphate.

(^2)
It may be argued that a cleavage-induced decrease in the affinity of ATP may not be reflected by a change in the K(0.5) of ATP for phosphoenzyme formation because of a concomitant cleavage-induced decrease in the rate of dephosphorylation. This is unlikely, however, because when the partially cleaved enzyme was phosphorylated by labeled ATP, and the disappearances of the phosphorylated alpha and 48-kDa peptides were examined over a 30-s period after addition of EDTA or unlabeled ATP, there was no significant difference between the two dephosphorylation rates.


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