(Received for publication, August 25, 1994; and in revised form, November 21, 1994)
From the
Cleavage of the -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
values of
ATP for phosphorylations of intact
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 of
ATP for protection of
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
+
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.
Controlled proteolysis of the -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.
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 ()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
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
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 [
C]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
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
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
[
C]ADP. The enzyme solubilized with
C
E
was prepared according to Esmann (16) and phosphorylated as the native enzyme but in reaction
mixtures containing 0.15% C
E
.
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 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
g for 10 min. The collected proteoliposomes from
several columns were pooled and centrifuged at 150,000
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 mM
NaCl, 0.2 mM EGTA, 10 µM valinomycin, 3 mM MgCl
, 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 mM
NaCl, 3 mM MgCl
, 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
and 100 mM Tris-HCl
(pH 7.2). The complete reaction mixture contained 10 mM
RbCl, 6 mM MgCl
, 5 mM P
, 1 mM ATP, and 100 mM Tris-HCl (pH
7.2). Reaction time was 1 min. Controls without P
and ATP
were included, and it was established that both P
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
was purified before use(12) .
Incubation of the enzyme with trypsin and K resulted in the cleavage of the
-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
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
after exposure to trypsin and K
. Enzyme samples
were exposed to trypsin for different periods. Remaining
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 -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
sequence(19) . This location is in agreement with that of the
cleavage point in the pig kidney enzyme(20) .
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
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
value of ATP for phosphorylation of the 48-kDa peptide was not
different from that of the intact
, indicating that the high
affinity ATP binding site is not disturbed by the cleavage. (
)
Figure 3:
Effects of varying ATP concentrations on
the phosphorylations of intact 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 -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 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
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
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
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
.
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
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
that have been shown to react with FITC(2) .
Figure 6:
Reaction of FITC with intact 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
and the 48-kDa peptide. Quantitation of the autoradiograms
of Fig. 7indicated equal sensitivities of the phosphorylations
of intact
and 48-kDa peptide to varying FITC concentrations. The K
value of FITC estimated from Fig. 7is
about the same as the K
value (5.7
µM) calculated previously from inactivation rate
constants(22) .
Figure 7:
Inhibitory effects of varying FITC
concentrations on Na-dependent phosphorylations of
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
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 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
-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
than with the 64-kDa peptide.
Figure 8:
Prevention of FITC reaction with 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
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
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
value of ATP for the protection of
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
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).
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
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
concentrations on phosphorylations of
and 48-kDa fragment (a) and inhibitory effects of AMPPCP on phosphorylations of
and 48-kDa fragment (b). In a, the partially
cleaved enzyme was reacted with
P
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
µM
P
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 phosphorylation(30) . In
experiments of Fig. 10b, the effects of AMPPCP on
phosphorylations of the intact
and 48-kDa peptide by P
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.
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) .
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 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.
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
/K
exchange (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
Na
for phosphorylation of the cleaved enzyme,
and its apparent K
affinity, are also not
significantly different from the corresponding Na
and K
affinities 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 K
access channels of E
(36) is also
disturbed by the cleavage.
The inability of the cleaved enzyme to
carry out P + 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
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
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
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.