From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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This paper extends our recent report on specific
iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of
E1 The molecular mechanism whereby Na,K-ATPase transduces the free
energy of hydrolysis of ATP into active transport of Na+
and K+ ions is unknown. We have a wealth of knowledge on
transport reactions, covalent phosphorylation,
E1/E2 conformational
transitions, and cation occlusion, embodied in the Post-Albers kinetic
mechanism (see Ref. 1). Active cation transport involves
Nacyt-dependent phosphorylation from ATP,
Na+ movement coupled to E1P Knowledge of the structural basis for energy transduction is meager due
to lack of information on molecular structure. The best structure of
Na,K-ATPase at 20-25-Å resolution reveals only the overall shape of
the protein and distribution of mass of Recently, we have described specific iron-catalyzed or copper-catalyzed
oxidative cleavage of renal Na,K-ATPase (13-15). The process seems to
involve a site-specific mechanism in which peptide bonds close to the
bound metals are cleaved, presumably by OH radicals generated locally
by the Fenton reaction or a reactive metal-peroxyl derivative (16-19).
Because more than one peptide bond can be cleaved from the same metal
site, this technique provides information on interacting segments of
individual subunits or neighboring subunits (unlike proteolytic
cleavage). Incubation of Na,K-ATPase with
Fe2+/ascorbate/H2O2 induces
specific cleavage of the Iron-catalyzed cleavages are very sensitive to the conformational state
(13). In (E2) or E2(Rb)
conformations, we observed four major and two minor fragments of the
This paper extends our observations on iron-catalyzed cleavages in two
ways. First, we have obtained further evidence for the site-specific
mechanism. Second, we have looked at iron-catalyzed cleavages in both
phosphorylated and non-phosphorylated conformations and effects of
inhibitors. The results provide novel information on the energy
transduction mechanism.
Na,K-ATPase, with specific activities of 12-17 units/mg
protein, was prepared from pig kidney (20) and was stored at Cleavage Reactions--
Suspensions of pig kidney Na,K-ATPase
(0.1-1 mg/ml) or rat microsomal preparations (1 mg/ml) were incubated
at 20 °C with freshly prepared solutions of 5 mM
ascorbate (Tris) plus 5 mM H2O2,
without or with added FeSO4. To arrest the reaction, 5-10 mM EDTA or 5-fold concentrated gel sample buffer with 5 mM EDTA was added. Samples were assayed for Na,K-ATPase
activity or applied to gels, respectively.
Gel Electrophoresis, Blotting to Polyvinylidene Difluoride
(PVDF)1, Immunoblots,
Sequencing--
Procedures for running of 7.5% Tricine SDS-PAGE,
including precautions prior to sequencing, electroblotting to PVDF
paper, immunoblots, and microsequencing of fragments have been
described in detail (22, 23).
Anti-K1012-Y1016, referred to as
"anti-KETYY," was used to detect fragments of the Calculations--
Non-linear curve-fitting was performed using
Enzfitter (Elsevier-Biosoft).
Materials--
For SDS-PAGE, all reagents were
electrophoresis-grade from Bio-Rad. Tris (ultra pure) was from Bio-Lab,
Jerusalem. L-(+)-ascorbic acid (catalogue no. 100127), 30%
H2O2 (catalogue no. 822287), and
Properties of Fe2+ Activation of Cleavage; Dependence
on E1/E2 Conformations--
Fig.
1 presents an immunoblot using anti-KETYY
to detect fragments produced in media containing different proportions
of K+ and Na+ ions (sum 150 mM). In
150 mM K+ (E2(K),
left), we observed five major fragments referred to as: a, near M1; b, at 214ESE;
c, near 283HFIH (previously referred to as near
IATL, see below); d, near 608MVTGD; and
e, at 712VNDS. Apparent
Mr values are 91.3, 80.6, 73.4, 38.2, and 26.3 kDa, respectively. By comparison with previous experiments done in low
ionic strength media (13), the cleavage near 367CSDK was
largely suppressed, while that near M1 was more prominent. About half
the
Bound Fe2+ ion is predicted to be in contact with more
residues in E2 than in E1
forms (see Ref. 13 and model in Fig. 11), and thus the Fe2+
should bind more tightly in E2 forms. Fig.
2 depicts the relationship between
Fe2+ ion concentration and cleavage of the
Fe2+ ions can replace Mg2+ ions in catalysis of
Na-dependent phosphorylation (25) and thus the question
arose whether Fe2+ ions bind to the Mg2+ site
and catalyze cleavage from this site. Fig.
3 depicts the cleavages at three
concentrations of Fe2+ (0, 1.5, and 15 µM
added in addition to the 0.05 µM contaminant in the
solutions), and Mg2+ from 0 to 7 mM. As the
Mg2+ ion concentration was raised, the cleavages at ESE,
near CSDK, near MVTGD, and at VNDS were progressively suppressed, while
cleavages near M1 and near HFIH were amplified (Fig. 3A).
Thus, Mg2+ ions stabilized an E1
conformation, noticeable especially at 7 mM. However,
quantification of amounts of the fragments, by scanning the
immunoblots, do not reveal any systematic influence of Fe2+
concentration on effects of Mg2+ ions, as seen in the
examples in Fig. 3B. Hence, Fe2+ and
Mg2+ ions do not compete and must bind at different
sites.
In Ref. 13, we speculated that histidine residues in the sequence HFIH
near M3 are involved in Fe2+ binding. To test this
hypothesis, and also the site-specific mechanism, we compared cleavage
of rat axolemma and rat kidney enzymes. Rat axolemma enzyme consists of
about 65% Effects of Phosphorylation, Inorganic Phosphate, Vanadate, and
Ouabain--
Fig. 5 depicts
iron-catalyzed cleavage of the pig kidney Na,K-ATPase in conditions of
Na-dependent phosphorylation from ATP. A low concentration
of ATP (5 µM) was used together with a regenerating system. In the presence of 140 mM Na+ ions, 0.5 mM Mg2+ ions, and the regenerating system, we
observed cleavages typical for the E1Na form (N
termini near M1 and HFIH). Upon addition of ATP and oligomycin, the
pump should be phosphorylated and, due to block of the
E1P
We calculated that, during incubation with
ATP/Mg2+/Na+/Rb+ in Fig. 5,
200-250 µM Pi could accumulate. Therefore,
we looked for effects on cleavages of Pi(Tris), 0.1-1
mM, without or with Mg2+ and Rb+
ions (Fig. 6). Addition of Pi
to a medium of low ionic strength indeed suppressed the cleavages near
CSDK and MVTGD, without significantly affecting other cleavages (Fig.
6A). In this condition, one could expect the enzyme to be in
an E2·P form, with Pi bound
non-covalently. In combination with 1 mM Mg2+
ions, addition of 2 mM Pi also partially
suppressed the cleavages at ESE and VNDS (Fig. 6B, see
legend for quantification based on scans). When added alone,
Mg2+ ions at 1 mM or lower concentrations had
little or no effect (see also Fig. 3). The presence of 1 mM
Rb+ ions did not alter the effect of Pi but
prevented partial suppression of cleavages at ESE and VNDS by the
combination of Pi and Mg2+ ions (Fig.
6C, see legend for quantification). Another effect of the
combination of Pi and Mg2+ ions was observed in
experiments that examined Pi concentration dependence of
suppression of the cleavage near MVTGD at 0-1 mM Mg2+. Data from scans of gels (Fig.
7) show that lower concentrations of
Pi were required in the presence of Mg2+ ions.
In the presence of 1 mM Mg2+ and 2 mM Pi, a substantial fraction of the enzyme
should be phosphorylated as a form referred to as
E2i-P, which is insensitive to
Rb+(K+) ions (31). In the presence of
Mg2+, Pi, and Rb+, a major fraction
should not be phosphorylated
(E2(Rb)·Pi) and a minor faction
could be phosphorylated in a Rb+(K+)-bound form
(E2-P·Rb) (31). An economical explanation of
the results in Figs. 5-7 is that either non-covalently bound phosphate or covalently bound phosphate, derived from ATP or Pi,
directly interfere with the cleavages near CSDK and MVTGD. In addition the cleavages at ESE and VNDS are somewhat suppressed in the
phosphorylated forms (see "Discussion" and Fig. 11).
Figs. 8 and
9 present some paradoxical observations.
As seen in Fig. 8, the presence of Mg2+/ouabain somewhat
suppressed cleavages near MVTGD and at VNDS, while in the presence of
Pi/Mg2+/ouabain all cleavages were suppressed
except the two near M1 and HFIH. Similarly, with vanadate (2 µM)/Mg2+, only the two cleavages near M1 and
HFIH were observed. Thus, the cleavage pattern with
Pi/Mg2+/ouabain and vanadate/Mg2+
was characteristic of E1 forms, although the
enzyme should be in an E2 form in both
conditions (see also Fig. 10). Fig. 9
presents another surprising finding with Pi. With the usual
order of addition of the components, first equilibration of enzyme with
Pi and then incubation with
Fe2+/ascorbate/H2O2, the effect of
Pi was as described in Fig. 6A. However, if the
enzyme was preincubated with both Pi and Fe2+
prior to addition of ascorbate/H2O2, the
cleavages at ESE and VNDS were also largely suppressed, although those
near M1 and HFIH were largely unchanged. The development of this effect
is seen in Fig. 9. After 20 min of preincubation of Pi with
Fe2+, prior to addition of
ascorbate/H2O2, the cleavage pattern was similar to that with Pi/Mg2+/ouabain or
vanadate/Mg2+.
Comparison of Chymotryptic and Iron-catalyzed Cleavages--
The
paradoxical results just presented led us to inquire whether the
expected conformations were indeed being stabilized in the different
conditions Well characterized chymotryptic cleavages (32) showed that
this was the case (Fig. 10). Thus, chymotryptic digestion in the
Na+-containing medium (E1Na) or in a
medium containing 7 mM Mg2+ ions
(E1Mg) produced the prominent fragment
"a," 74.6 kDa, while in the Rb+-containing
medium (E2(Rb)) "a" was
suppressed and "b" and "c" were more
prominent. Again, as expected in the medium containing ouabain/Mg/Pi (E2-P·ouabain),
"b" and "c" were major and
"a" was minor. An incidental benefit was that defined
chymotryptic fragments allowed better determination of the positions of
two iron-catalyzed cleavage fragments, which could not be sequenced due
to blocked N termini. The N terminus of fragment "a" is
Ala267 (33), and sequencing of fragments "b"
and "c" showed that the N termini are Val440
and Ala591, respectively. The apparent
Mr values of "a" and
"c" 74.6 and 39.3 kDa are 1.2 and 1.1 kDa greater than
those of the closest iron-catalyzed cleavage fragments, 73.4 and 38.2 kDa respectively. Therefore, the N termini of the latter are
approximately 10 residues downstream, i.e. they are near
283HFIH and 608MVTGD.
Fig. 11 depicts schematically the
proposed arrangement of the peptide sequences around the bound
Fe2+ ion in different states. Table
I summarizes the specificity of the
different cleavages and also gives a rough measure of their prominence,
which reflects presumably proximity to bound Fe2+ ions.
E2
conformational transitions (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to
a bound Fe2+ ion are cleaved, and provide evidence on
proximity of the different cleavage positions in the native enzyme. A
sequence HFIH near trans-membrane segment M3 appears to
be involved in Fe2+ binding. Previously we hypothesized
that E2 and E1
conformations are characterized by formation or relaxation of
interactions within the
subunit at or near highly conserved
sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and
VNDSPALKK in the major cytoplasmic loop. This concept has been tested
by examining iron-catalyzed cleavage in both non-phosphorylated and
phosphorylated conformations and effects of phosphate, vanadate, and
ouabain. The results imply that both E1
E2 and E1P
E2P transitions are indeed associated with
formation and relaxation of interactions between cytoplasmic domains,
comprising the minor loop plus N-terminal tail leading into M1 and
major loop, respectively. Furthermore, it appears that either
non-covalently or covalently bound phosphate bind near CSDK and MVTGD,
and Mg2+ ions may bind to residues within TGES and
VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions
seem to occur within or near the active site. We discuss the
relationship between structural changes in the cytoplasmic domain and
movements of trans-membrane segments that lead to cation transport.
Presumably conformation-dependent formation and relaxation of
domain interactions underlie energy transduction in all P-type pumps.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E2P, Kexc-activated
dephosphorylation, K+ movement coupled to
E2(K)
E1.
and
subunits (2).
Recent cryoelectron microscopy studies of Ca-ATPase and H-ATPase
demonstrate the overall shape at 8-Å resolution, including "head",
"neck," and membrane sectors, including 10 trans-membrane
-helical rods, most of which are tilted at an angle to the membrane
(3, 4). The topological organization with 10 trans-membrane segments
confirms that shown by a variety of techniques (5). Biochemical and
molecular techniques are providing much information on residues
involved in cation occlusion within trans-membrane segments (6),
primarily M4, M5, and M6 (7-9), or ATP binding within the large
cytoplasmic loop (see Ref. 5). These studies indicate the necessity for
interactions between the ATP sites and cation occlusion sites. These
interactions are mediated by E1
E2 conformational transitions, which have been
studied extensively using proteolytic digestion, fluorescent probes,
ligand binding, etc. (see Ref. 10 for a review and references). The
fact that probes bound at different sites report the
E1
E2 transition
implies that substantial structural changes must occur. However, the
nature of those changes has been largely obscure. All P-type pumps
contain the conserved cytoplasmic sequences TGES in the minor loop
between M2 and M3, MVTGD in the major loop, and TGDGVNDSPALKK in the
so-called "hinge region" before M5 (5). Proteolytic cleavage and
site-directed mutagenesis in these sequences usually stabilize
E1 conformations, implying an involvement in the
conformational transitions (see Refs. 5 and 8 for full references).
Based on functional effects of mutations and proteolytic cleavages in
the
-strand minor cytoplasmic loop of sarcoplasmic reticulum
Ca-ATPase, an interaction between the minor and major cytoplasmic loop
near the phosphorylation site was proposed earlier (11). For yeast
H-ATPase, mutations within the minor loop suggested a similar
conclusion (12).
subunit at the cytoplasmic surface without
cleaving the
subunit (13). Copper-catalyzed oxidative cleavage
occurs at the extracellular surface and both
and
subunits are
cleaved (15).
subunit. In E1 or E1Na conformations, cleavage was much slower and
only one major cleavage was observed. Positions of cleavages were
either identified exactly by N-terminal sequencing or, for fragments
with blocked N termini, approximately using sequence-specific
antibodies. In E2 conformations only, cleavages
(identified by short sequences at or near the N termini) were found at
214ESE in the minor loop between M2 and M3, near
367CSDK after M4 which includes the phosphorylated
Asp369, near 608MVTGD in the major loop, and at
712VNDS in the "hinge" region before M5. In either
E2 or E1 conformations, a
cleavage was seen near 263IATL, before M3. The observations
suggested strongly that peptide bonds are cleaved with a probability
depending on their proximity to a bound Fe3+ or
Fe2+ ion, and thus imply that the different cleavage points
are also in proximity to each other. Several cleavages lie in or near
the highly conserved cytoplasmic sequences, suggesting that these sequences mediate mutual interactions. We proposed that, in
E2 conformations, the minor and major loops
interact near the conserved sequences while, in
E1 conformations, the loops separate
(13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C
in a solution of 250 mM sucrose, 25 mM
histidine, pH 7.2, and 1 mM EDTA (Tris). Rat axolemma
microsomes (2-3 units/mg enzyme) prepared as in Ref. 21 or rat kidney
microsomes (3-4 units/mg protein) were prepared as in Ref. 20. Prior
to incubation with Fe2+/ascorbate/H2O2, the microsomal
membrane preparations (1.5 mg/ml) were pretreated with sodium
deoxycholate 1.2 mg/ml for 15 min at 20 °C, washed twice, and
resuspended in a solution containing 10 mM Tris·HCl, pH
7.2.
subunit.
Immunoblots were stained with diaminobenzidine with metal ion
enhancement (15-20 µg of protein/lane) (24) or developed by enhanced
chemiluminescence (ECL; 3-5 µg of protein/lane) using anti-rabbit
IgG horseradish peroxidase conjugate and the protocol supplied with ECL
reagents from the 1998 Amersham Pharmacia Biotech catalogue. For
quantification of bands, the stained PVDF paper or developed x-ray
films were scanned with a Bio-Rad GS-690 imaging densitometer and
analyzed with Bio-Rad Multi-Analyst software (version 1.01). For
quantification of Coomassie-stained
subunit the band was cut out of
the gel, and optical density of Coomassie stain extracted into 1% SDS
solution was measured at 595 nm (13).
-chymotrypsin (catalogue no. 2307) were from Merck. Phosphocreatine, P6502, creatine phosphokinase P3755. and oligomycin O4876 were from
Sigma. All other reagents were of analytical grade.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit was cleaved in these conditions. As K+ was
replaced by Na+ ions, cleavage at ESE, near MVTGD, and at
VNDS was progressively suppressed, essentially completely at 150 mM Na+ (E1Na,
right), while in parallel, the cleavages near M1 and HFIH were amplified. A similar change of pattern was observed upon transfer
of the enzyme from low ionic strength (10 mM Tris, pH 7, E2) to high ionic strength media (10 mM Tris, pH 7, 300 mM choline
chloride, E1).
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Fig. 1.
Iron-catalyzed cleavage of the
subunit at different K+ and
Na+ concentrations. Na,K-ATPase (1 mg/ml) was
suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and 150 mM of KCl plus NaCl in the indicated proportions.
10 µM FeSO4 was added and then 5 mM ascorbate and 5 mM
H2O2; after 2 min of incubation at 20 °C,
the reaction was stopped with concentrated gel buffer containing 5 mM EDTA and samples were applied to the gel.
subunit and
inactivation of Na,K-ATPase activity, in Rb+- and
Na+-containing media (E2(Rb) or
E1Na, respectively). The curves are fitted well
by simple hyperbolas with K0.5 values of 0.57 and 3.49 µM for cleavage of the
subunit and 0.20 and
1.16 µM for inactivation of Na,K-ATPase, respectively. As
discussed previously (13, 15), the 2-3-fold lower
K0.5 value for inactivation of Na,K-ATPase
compared with cleavage could imply that oxidative reactions occur in
addition to chain cleavage and inactivate the enzyme. In any event, the
5-6-fold lower values of K0.5 in the E2(Rb) compared with the
E1Na conformation, by either measure, are
compatible with the prediction.
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Fig. 2.
Fe2+ concentration dependence of
cleavage of the subunit and inactivation of
Na,K-ATPase activity in Rb- or Na+-containing
media. Na,K-ATPase (0.1 mg/ml) suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and 30 mM RbCl or NaCl,
and was incubated with Fe2+ at varying concentrations and 5 mM ascorbate and H2O2 for 2 min at
20 °C. Samples were either taken for determination of the
Na,K-ATPase activity or applied to a gel (45 µg of protein/lane), and
the
subunit was quantified as described under "Experimental
Procedures."
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Fig. 3.
Iron-catalyzed cleavage of the
subunit at different Fe2+ and
Mg2+ concentrations. A, Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mM
Tris·HCl, pH 7.2, and MgCl2 (0, 0.15, 1.5, and 7 mM, respectively). Each of the four enzyme suspensions were
divided into three aliquots, and 0, 1.5, or 15 µM
FeSO4 was added together with 5 mM
ascorbate/H2O2. The incubation time without or
with added Fe2+ was 15 or 2 min respectively. B,
diaminobenzidine-stained PVDF paper was scanned and analyzed as
described under "Experimental Procedures." The figures depict the
quantities of the indicated fragments at the different Mg2+
concentration as a percentage of the quantity of the fragment in the
absence of Mg2+ ions.
3, 25%
2, and only 10%
1 isoforms, while kidney
enzyme is essentially all
1 isoform (26-28). In
3 and
2
isoforms, the second histidine is replaced by a glutamine,
i.e. HFIQ. The experiment in Fig. 4A compared the time course of
cleavage of axolemma and kidney enzymes. For axolemma enzyme, the
cleavage near HFIH was largely absent and that at VNDS was less
prominent, while the cleavages at ESE and near MVTGD were similar to
those of the kidney enzyme. For axolemma, a small amount of the
fragment with N terminus near CSDK also appeared, although this is less
certain because the control itself contains a minor fragment with the
same mobility. (The axolemma fragments cleaved near MVTGD and at VNDS
have slightly lower mobility than equivalent fragments of kidney (41.8 versus 39.1 and 29 versus 27 kDa, respectively).
However, this cannot be taken to indicate that the cleavage sites are
different, since intact
3 and
2 also have a slightly lower
mobility than the
1 isoform, even though the
Mr value of rat
1 (112, 566) is slightly higher than
2 (111, 580) or
3 (111, 735) (27, 29).) Fig. 4B depicts cleavage of axolemma enzyme in Rb+-
or Na+-containing media at different concentrations of
added Fe2+ ions. The two major fragments, with N termini
ESE and near MVTGD, increased in amount as Fe2+ was raised
progressively to 10 µM, as did the minor fragments. In
parallel experiments with axolemma and kidney enzyme (data not shown),
it was found that cleavage of axolemma enzyme required significantly
higher concentrations of added Fe2+ ions. For example, the
K0.5 values for appearance of the fragment with
N terminus near MVTGD were 1 ± 0.2 µM for kidney
and 1.95 ± 0.2 µM for axolemma, respectively. In
the sodium-containing medium, cleavage of both the axolemma and kidney
enzymes was suppressed and the fragment with N terminus ESE and the
fragment near MVTGD were not observed. In summary, axolemma and kidney
enzymes differ in specificity of the cleavages and affinity for
Fe2+ ions, whereas the E2(Rb)
E1Na transition affects cleavage essentially similarly.
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Fig. 4.
Iron-catalyzed cleavage of rat axolemma and
kidney Na,K-ATPase. A, deoxycholate-treated rat
axolemma (2.1 units/mg) or kidney (4.3 units/mg) microsomes were
suspended at 1 mg/ml in a medium containing 10 mM
Tris·HCl, pH 7.2, and 30 mM RbCl and incubated for the
indicated times with Fe2+ 0.15 µM, plus 5 mM ascorbate/H2O2. The axolemma or
kidney microsomes were applied to a gel (25 or 12.5 µg of
protein/lane respectively, in order to make equal the amount of subunit in each case). B, deoxycholate-treated axolemma
microsomes (1 mg/ml), suspended in media containing 30 mM
RbCl or NaCl, were incubated with the indicated concentrations of
FeSO4 and 5 mM
ascorbate/H2O2 for 5 min. 25 µg of protein
was applied per lane.
E2P
conformational transition (30), the predominant form should be
E1P. This condition indeed produced cleavages
typical of an E1 conformation. In the absence of
oligomycin, the combination of ATP/Na+/Mg2+
phosphorylates the pump, but the predominant form should now be
E2P. In this condition, we observed two
additional cleavages typical of an E2 form (N
termini ESE and VNDS), but, strikingly, the prominent cleavage near
MVTGD normally seen for unphosphorylated E2 or
E2(K) forms did not appear. The cleavage near
CSDK was not seen, but this was expected in the high ionic strength
medium. Addition of a low concentration of Rb+ (2 mM) to the medium containing
ATP/Na+/Mg2+ accelerates dephosphorylation from
high affinity extracellular sites, leading to
E2(Rb) as the predominant form. In this
condition, the fragments with N termini ESE and VNDS were somewhat
amplified, but again the cleavage near MVTGD did not appear. The latter
result was surprising because the cleavage near MVTGD is very prominent in the E2(Rb) form generated directly by adding
Rb+ (K+) ions to the enzyme (see Ref. 13 and
this paper). The cleavage pattern in the medium containing 2 mM Rb+, 150 mM Na+, and
0.5 mM Mg2+, without ATP, shows that the enzyme
remained in the E1 form. Thus, suppression of
the cleavage near MVTGD in the presence of Rb+ ions and
ATP/Na+/Mg2+ was associated with formation of
the E2(Rb) form via dephosphorylation of
E2P. It seemed possible that ATP itself or the
products of its hydrolysis, ADP and Pi, were responsible
for suppressing the cleavage near MVTGD, and thus a number of control
experiments were performed. In low ionic strength media
(E2), the cleavage near MVTGD was prominent, but
neither ATP nor ADP at 5 µM had any effect (data not
shown).
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Fig. 5.
Iron-catalyzed cleavage of the
subunit in phosphorylated and dephosphorylated
conformations. Na,K-ATPase (0.6 mg/ml) was suspended at 20 °C
in a medium containing 10 mM Tris·HCl, pH 7.2, 140 mM NaCl, 2 mM creatine phosphate (sodium salt),
creatine phosphokinase (48 units/ml) and where indicated 0.5 mM MgCl2, 5 µM ATP(Tris), 150 µg/ml oligomycin, and 2 mM RbCl. The samples were then
incubated with 5 µM FeSO4 and 5 mM ascorbate/H2O2 for 2 min at
20 °C.
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Fig. 6.
Effects of Pi, Mg2+,
and Rb+ ions on iron-catalyzed cleavage of the
subunit. Na,K-ATPase (1 mg/ml) was suspended
in a medium containing 5 mM Tris·HCl, pH 7.2, and 0, 0.1 or 1 mM Pi(Tris) (A) or 1 mM MgCl2 and 2 mM Pi as
indicated in B or 1 mM MgCl2, 2 mM Pi, and 1 mM RbCl as indicated
in C, and incubated with 5 µM
FeSO4 and 5 mM
ascorbate/H2O2 for 2 min at 20 °C. Amounts
of fragments in arbitrary units based on scans of immunoblots:
B, ESE: control, 7.2; Mg, 7.0; Pi, 6.0;
Pi/Mg, 4.9; VNDS: control, 6.26; Mg, 6.0; Pi,
6.7;Pi/Mg, 4.4. C, ESE: control, 7.2; Mg, 7.3;
Pi, 7.1; Pi/Mg, 5.9; VNDS: control, 6; Mg, 5.9;
Pi, 6.3; Pi/Mg, 6.2.
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Fig. 7.
Co-operative effect of Pi and
Mg2+ ions in suppression of the specific cleavage near
MVTGD. Na,K-ATPase (1 mg/ml) was suspended in a medium containing
10 mM Tris·HCl, pH 7.2, and 0, 0.1, 1, or 5 mM Pi(Tris) and 0, 0.1, or 1 mM
MgCl2. The ionic strength was maintained constant by
addition of choline chloride as necessary. The suspension was incubated
with 5 µM FeSO4 and 5 mM
ascorbate/H2O2 for 2 min at 20 °C. The
diaminobenzidine-stained PVDF paper was scanned and analyzed as
described under "Experimental Procedures." The figure depicts the
quantity of the fragment near MVTGD at different Pi
concentrations as a percentage of that without Pi, at the
MgCl2 concentration of 0, 0.1, and 1 mM.
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Fig. 8.
Effect of
Pi/Mg2+/ouabain or vanadate/Mg2+ on
iron-catalyzed cleavage of the subunit.
Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mM Tris·HCl, pH 7.2, 1 mM MgCl2,
and 2 mM Pi and 1 mM ouabain as
indicated (A), or 0.5 mM MgCl2 and 2 µM vanadate (Tris) as indicated (B), and
incubated with 10 µM FeSO4 and 5 mM ascorbate/H2O2 for 1 min at
20 °C.
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Fig. 9.
Effect of preincubation of Pi
with Fe2+ ions on iron-catalyzed cleavage of the
subunit. Na,K-ATPase (1 mg/ml) was suspended
in a medium containing 10 mM Tris·HCl, pH 7.2, 1 mM Pi (Tris), and 5 µM
FeSO4 and was incubated for 0-20 min. Then 5 mM ascorbate/H2O2 was added and the
suspension incubated for 2 min at 20 °C.
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Fig. 10.
Chymotryptic digestion of Na,K-ATPase.
Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mM Tris·HCl, pH 7.2, 20 mM NaCl, or 20 mM RbCl or 7 mM MgCl2
(A) or 1 mM Pi, 1 mM
MgCl2, and 1 mM ouabain (B).
-Chymotrypsin was added at a ratio of 1:20 (w/w) with respect to
Na,K-ATPase and incubated together at 37 °C for 10 min. 150 mM RbCl was added and then 1 mM PMSF, and the
mixture was incubated for 15 min at room temperature before
centrifugation to remove the chymotrypsin, and solubilization of the
pellet in the gel buffer. In A, 25 µg of protein were
applied per lane (diaminobenzidine stain), and, in B, 5 µg
were applied per lane (ECL).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Schematic drawings of interactions between
bound Fe2+ ions and the Na,K-ATPase in different
conformational states.
Fragments observed in different conformational states
The Fe2+ Binding Site--
Previously, we proposed the
site-specific mechanism in which each subunit is cleaved at only
one of the different points of contact between the polypeptide chain
and a bound Fe2+ ion, as depicted in Fig. 11 (13, 14). The
additional observations discussed here support this notion and
essentially exclude the possibility that cleavages are catalyzed by
Fe2+ bound at several sites. One might consider a
hypothesis that cleavages occur at two Fe2+ sites, one of
which includes the points near M1 and HFIH and does not change in
E1 and E2 forms, while
the other includes the points at ESE, near CSDK, near MVTGD and at VNDS
and exists only in E2 forms. Although it is
difficult to rigorously exclude this idea, the following arguments
favor one Fe2+ site.
1) Parallel suppression of cleavages at ESE, near MVTGD and at VNDS and amplification of cleavages near M1 and HFIH, upon transition from E2(K) to E1Na (Fig. 1) or other E1 forms (Fig. 3), is explained most simply by assuming that the contacts at ESE, near MVTGD and at VNDS move away while those near M1 and HFIH remain near the bound Fe2+ ion. Accordingly, the probability of cleaving either decreases or increases respectively.
2) In Fig. 2, the curves fit well to simple hyperbolae with higher
apparent affinities for inactivation of Na,K-ATPase or cleavage in a
Rb+-containing as opposed to a Na+-containing
medium (K0.5 of 0.2 or 0.57 versus
1.16 or 3.49 µM, respectively), consistent with
Fe2+ binding to a single site. Apparent free energies of
Fe2+ binding, calculated from the
K0.5 for inactivation or cleavage, in
Rb+- compared with Na+-containing media, are
9.02 or
8.43 versus
8.01 or
7.36 kcal/mol, respectively, giving a difference of about
1 kcal/mol by either measure. Assuming that the different cleavages between the
E2(Rb) and E1Na
conformations are catalyzed at a separate Fe2+ site, the
K0.5 for Fe2+ at this site would
correspond to the
1 kcal/mol or 0.18 M, i.e. an unrealistic value that argues strongly against the idea of two
sites. In contrast, the data imply that most of the binding energy for
Fe2+ comes from residues that bind in either
E2 or E1 forms
(e.g. HFIH), while the extra contacts in
E2 forms are energetically weak.2
3) Lack of competition between Mg2+ and Fe2+ ions (Fig. 3) shows that Mg2+ and Fe2+ bind at different sites. Other data discussed below (see also Fig. 11) suggest that, in the presence of phosphate, Mg2+ ions can interact with the protein near the cleavage sites at ESE and VNDS. These observations also favor the notion of one Fe2+ site rather than two separate sites, neither of which recognize Mg2+ ions.
4) Although there are significant differences in sequences of the rat
3,
2, and
1 isoforms (85-86% identity; Ref. 29), it is
likely that the different cleavage patterns of axolemma (mainly
3
and
2) and kidney (
1) enzyme (Fig. 3) are attributable to the
His286
Gln substitution. First, the cleavage near HFIH
is essentially absent in axolemma enzyme. Second, the fact that
cleavages, at ESE and near MVTGD, occur in both axolemma and kidney
enzymes and undergo a similar response to the
E2(Rb)
E1 transition
(Fig. 4B) indicates that spatial organization of the
3
and
1 isoforms is similar. Thus, reduced cleavage at VNDS in
addition to that at HFIH suggests that the Gln for His substitution
alters the disposition of both VNDS and HFIH next to the bound
Fe2+ ions and weakens Fe2+ binding. Conversely,
reduced cleavage at VNDS catalyzed at a separate Fe2+ site
is a less likely possibility since the sequences of
1,
2, and
3 isoforms are identical over a long stretch either side of VNDS.
Unequivocal evidence for involvement of histidine residues will require
site-directed mutagenesis.
5) Treatment of renal Na,K-ATPase with the histidine-specific reagent diethylpyrocarbonate prevents all iron-catalyzed cleavages, clearly implicating histidine residues.3 Histidines are found only at the cleavage site near HFIH and MVTGDH.
Effects of Phosphorylation, Inorganic Phosphate, Vanadate, and Ouabain-- Two salient features in Fig. 5 are as follows: 1) in E1P (ATP/Na+/Mg2+/oligomycin), the cleavage pattern is typical for E1; 2) in E2P (ATP/Na+/Mg2+), two cleavages typical of E2 or E2(Rb) appear (at ESE and VNDS), if less prominently than in the non-phosphorylated form (ATP/Na+/Mg2+/Rb+), but the major cleavage near MVTGD is not observed. Fig. 6 clarifies the difference between the phosphorylated or unphosphorylated E2 forms by showing the following. 1) Non-covalent binding of Pi in E2·P selectively suppresses the cleavages near CSDK and MVTGD (Fig. 6A). An equivalent form is achieved in the presence of Pi/Mg2+/Rb+ (Fig. 6C), or ATP/Na+/Mg2+/Rb+ due to ATP hydrolysis (Fig. 5). 2) In E2i-P (Pi, Mg2+), the pattern is the same as in E2-P (Fig. 6B). E2P and E2i-P represent different subconformations, as indicated by different responses to alkali cations, methyl hydroxylamine, and vanadate (31, 34, 35). Since their iron-cleavage pattern is the same, presumably the difference between E2P and E2i-P is restricted to the microenvironment of the bound phosphate. On the basis of Figs. 5 and 6, we propose that either non-covalently bound (E2·P shown in Fig. 11) or covalently bound phosphate (E2-P, E2i-P) interacts directly with residues near CSDK and MVTGD, hindering access of the bound Fe2+ and cleavage.
Mg2+ ions are tightly bound to the phosphoenzyme formed from either ATP (E2-P) or Pi (E2i-P) (31, 25, 36). In order to explain the less prominent cleavages at ESE and VNDS in the E2-P·Mg complex compared with non-phosphorylated forms (seen in both Figs. 5 and 6), and the synergism between Pi and Mg2+ ions in suppressing cleavage near MVTGD (Fig. 7), we propose that Mg2+ ions are bound near ESE and VNDS and also to the bound phosphate. In this way, access of the Fe2+ to the sites at ESE and VNDS may be reduced. In the presence of Pi, Mg2+ ions, and ouabain (Fig. 8), ouabain may induce the Mg2+ ions in the E2-P·Mg complex to bind to its contact residues more tightly or in such a way as to completely prevent access of the bound Fe2+ ions and cleavage at ESE, near CSDK and MVTGD, and at VNDS (Fig. 11). Similarly vanadate may bind near CSDK and MVTGD with Mg2+ ions bound near ESE and VNDS and to the vanadate itself, suppressing cleavages at ESE, near CSDK and MVTGD, and at VNDS (Fig. 11). Thus, the paradox that Pi/Mg2+/ouabain or vanadate/Mg2+ give E1-like cleavage patterns, although these ligands stabilize E2 forms, is explained by assuming that the bound ligands directly hinder access of the bound Fe2+ ions to the cleavage sites. Finally, the paradox (Fig. 9) that preincubation of Pi with Fe2+ leads to a pattern similar to that with Pi/Mg2+/ouabain or vanadate/Mg2+ may imply that, Fe2+ ions slowly gain access to normal Mg2+ binding sites (see Ref. 25) to produce a complex with the Pi. In this complex with Pi, the Fe2+ is itself redox-inactive, but access of the redox active Fe2+ bound near HFIH (Fig. 11) to the cleavage sites is hindered.
Although our findings do not identify precisely co-ordinating residues
for Mg2+ ions and phosphate, some reasonable suggestions
can be made. Based on a structural analogy to EF hand proteins and
cation binding to synthetic peptides, it was proposed that
Mg2+ ions are co-ordinated with Asp707 in the
sequence EITAMTGDVN707DSPALKK of Ca-ATPase. (37). Studies
of O18 exchange between Pi and water after
mutagenesis of Asp586 of Na,K-ATPase, and sequence and
structural homologies with adenylate kinase, suggest that conserved
sequences 586DPPR and 608MVTGDHPITAK may be
involved in co-ordination of Mg2+ ions and the
-phosphate group of ATP (38, 39). Proximity of the
-phosphate of
ATP to the sequence VNDS can be inferred from covalent labeling at
Asp710 (40) and at Lys719 (41).
Mg2+ ions could be co-ordinated to several residues, such
as Asp714 and Glu214 at VNDS and ESE as
depicted in Fig. 11. Non-covalently bound phosphate could interact with
Lys370 in CSDK, and both non-covalently and covalently
bound phosphate could also interact with Lys618 near MVTGD
(or Arg589 in the DPPR sequence).
The effect of Pi alone (Fig. 6) and synergism between Pi and Mg2+ (Fig. 7) indicate that Pi and Mg2+ bind in an unordered but positively co-operative fashion (see also Refs. 36 and 42). Our findings do not agree with a proposal that Pi binds after Mg2+ ions (38).
Implications for the Energy Transduction Mechanism-- By comparison with our initial study of iron-catalyzed cleavages, several additional conclusions emerge.
1) Characterization of E2(K) and E1Na forms by formation or relaxation of interactions between minor and major cytoplasmic loops can be modified slightly to include a role of the N-terminal cytoplasmic segment. Parallel behavior of the cleavages near M1 and M3 (HFIH) implies proximity between M1 and M3 and interactions between the segment leading into M1 and loop between M2 and M3 (probably via salt bridges; Ref. 33). Proteolytic cleavage or truncated mutants in the N-terminal segment shift the conformational equilibrium toward E1 (33, 43), as do proteolytic cleavages or mutations in the loop between M2 an M3 (Refs. 32 and 44; see Ref. 5 for references to other P-type pumps). Recently, an interaction between the cytoplasmic tail and the loop between M2 and M3 and possibly with the large cytoplasmic loop (45) has been inferred on the basis of strong synergism in the functional effects of a double mutant (with a truncation at residue 32 (a1M32) and also G233K or G233Q mutation, referred to as a1M32G233K or a1M32G233KQ). Our results fit this interpretation assuming that the N-terminal segment together with the minor loop constitute one domain and the major loop a second domain.
2) Formation or relaxation of the domain interactions in
E2(K) and E1Na are
paralleled in the phosphorylated conformations E2P and
E1P and, we suggest, constitute an essential
element of energy transduction. These interactions seem to occur within
or near the active site, so explaining interference with cleavages of
non-covalently or covalently bound phosphate or vanadate (Fig. 11). A
change of microenvironment of bound phosphate from hydrophilic in high
energy E1P to hydrophobic in low energy
E2P has been proposed based on effects of
organic solvents (46). The aqueous or less aqueous microenvironment of
bound phosphate in E1P or
E2P could be explained by the open or closed
domain structure, respectively. A Mg2+ ion is tightly bound
in E2P, and it can be proposed that tight co-ordination of the Mg2+ ion near TGES and VNDS and to the
covalently bound phosphate induces the interactions between the minor
and major loops which underlies the E1P
E2P transition. In E2P,
the bound phosphate (at CSDK and MVTGD) and the interacting sequences
of the minor and major loops (TGES and VNDS) must lie fairly close to
the membrane in order to explain proximity to HFIH near the entrance to
M3. Accordingly, the high affinity ATP site in
E1 should be organized such that the
-phosphate of ATP interacts near CSDK and MVTGD, while the purine
binding residues (such as Lys501 and Gly502
(47, 48) and Lys480 (49)) are located toward the periphery
of the major cytoplasmic loop.
3) Cation transport involves deocclusion of Na+ or
K+ ions accompanying E1P
E2P and E2(K)
E1, respectively, and requires opening or
closing of "gates," which act as barriers to dissociation of cations. "Gates" could be formed by interacting residues of
adjacent trans-membrane segments. In E1P(3Na)
and E2(2K), "gates" are closed at both
surfaces. In E2P, an extracellular "gate" is
open and a cytoplasmic "gate" is closed, while in
E1 the cytoplasmic "gate" is open and an
extracellular "gate" is closed. We propose that formation or
relaxation of interactions between cytoplasmic domains alters the twist
or tilt or perhaps stretch of the relevant trans-membrane helices and
so alters their mutual interactions, i.e. the "gates." Prime candidates as mobile trans-membrane segments are M4, M5, and M6,
which contain residues involved in cation occlusion (7-9). Movement of
M5, particularly Asn776, Ser775,
Thr774, Thr771, and Tyr771 in the
sequence YTLTSNIPEIT, seems to be important in the connection between
the major loop and the membrane domain (50), as is Leu332
in the sequence 328PEGLL near the cytoplasmic boundary of
M4 (51). There is evidence that M9 is a mobile segment, although the
role of M7-M10 in cation occlusion is unclear (52). M1-M3 do not seem
to play a role in cation occlusion, and indeed the lack of
conformational sensitivity of the cleavages near M1 and M3 (HFIH)
implies that M1 and M3 are static. The coupling mechanism described
above is similar to a "scissors mechanism" proposed by Jørgensen
et al. (53) with one significant difference. In Ref. 53, the
handles of the scissors are represented by the sequences near
Asp369 and Asp714 within the major cytoplasmic
loop, which interact via the bound phosphate and Mg2+ ions.
On our model, the handles represent the interacting minor and major
loops as depicted in Ref. 13 and in Fig. 11. In both cases, the
trans-membrane segments represent the blades of the scissors.
Conclusion--
Because the sequences involved in cytoplasmic
domain interactions are highly conserved, the mechanism of energy
coupling proposed here should apply to all P-type pumps. In this
respect, it is remarkable that recent cryoelectron microscope images of
Ca-ATPase indeed reveal a compact cytoplasmic domain in the
E2 form but an open structure in the
E1 form, with distinct lobes separated by a deep
gorge (54).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. Kyte (University of California, San Diego, CA) for providing anti-KETYY and to Dr. R. Blostein (McGill University, Montreal, Canada) for providing rat axolemma and rat kidney microsomes.
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FOOTNOTES |
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* This work was supported by a grant from the Israel Science Foundation.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.: 972-8-934-2278;
Fax: 972-8-934-4118; E-mail:
bckarlis{at}weizmann.weizmann.ac.il.
2 If a fraction of added Fe2+ is bound to the membranes, ascorbate, etc., the true binding affinity of Fe2+ will be higher than the calculated affinity, but the relative difference in Rb+- versus Na+-containing media will remain. This possibility can only strengthen the argument against two Fe2+ sites.
3 D. Tal, J. Capasso, and S. J. D. Karlish, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine.
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REFERENCES |
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