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(Received for publication, November 27, 1995; and in revised form, January 31, 1996) From the
Treatment of purified canine renal Na,K-ATPase with a range of
photoactivatable amiloride derivatives results in inhibition of ATPase
activity prior to illumination. Inhibition by amiloride derivatives
substituted on a guanidium N could not be prevented by the presence of
either K or Na; however, these cations could protect the enzyme against
inhibition by derivatives substituted on the 5-position of the pyrazine
ring. In the case of
5-(N-ethyl-[2`-methoxy-4`-nitrobenzyl])amiloride
(NENMBA), the presence of monovalent cations (Na, K, and Rb) protected
the enzyme effectively against inhibition, with concentrations in the
millimolar range. ATP did not prevent inhibition; furthermore, native
and NENMBA-treated enzyme exhibited normal levels of high affinity
[
The sodium pump or Na,K-ATPase (EC 3.6.1.37) is the plasma
membrane protein responsible for maintaining the resting concentrations
of sodium and potassium in animal cells. It is composed of an
A detailed molecular
description of these transport events includes the localization of
which amino acid residues are involved in binding and transport of
sodium and potassium across the cell membrane. One method that has been
used to address this problem is the utilization of residue-selective
chemical probes to modify purified sodium pump protein (for review, see (13) ). It has been assumed that negatively charged amino acids
are involved in cation coordination. This hypothesis has been tested by
using aspartate and glutamate-specific carbodiimides which inactivate
the sodium pump in a cation-preventable
manner(14, 15, 16, 17, 18, 19, 20, 21) .
There are inherent ambiguities in the use of such reagents, as they can
also induce the formation of endo-peptide bonds; such crossing linking
is also cation-preventable(16, 19) . In order to
circumvent these problems, 4-(diazomethyl)-7-(diethylamino)-coumarin
(DEAC), ( In this report we have examined the
effects of four photoactivatable amiloride derivatives on the
Na,K-ATPase. Amiloride derivatives were chosen because they are known
to inhibit other sodium transporting proteins such as epithelial Na
channels, the Na/H antiporter, and the Na/Ca
exchanger(24, 25) . Furthermore, there are well
defined structure-activity relationships for amiloride derivatives;
guanidium-substituted probes show high affinity for epithelial Na
channels, whereas pyrazine ring derivatives evidence a higher affinity
for exchangers than channels(25) . The four compounds we tested
are representative of the two classes of inhibitors. All four reagents
inhibit the Na pump, but only the pyrazine ring-substituted derivatives
in a cation-preventable manner. The most effective of these probes,
NENMBA (Fig. 1), was shown to be covalently incorporated into
the
Figure 1:
Chemical structures of the four
photoactivatable, amiloride derivatives.
Figure 2:
Summary of inhibition of purified
Na,K-ATPase by the amiloride derivatives. The enzyme (0.05 mg/ml) was
incubated with each compound (100 µM) at 37 °C for 2
min ± Rb, K (10 mM) or Na (60 mM, where
applicable) at pH 7.5 (40 mM Hepes) and activity was assayed
as described under ``Materials and
Methods.''
Figure 3:
Effect of varying [NENMBA] on
the time course of Na,K-ATPase activity. The enzyme (0.05 mg/ml) was
incubated with 20 µM (
Figure 4:
Protective effect of Rb (A) and
Na (B) on the inhibition of purified Na,K-ATPase by NENMBA (Rb
is used as a cogener for K in these experiments). The enzyme (0.05
mg/ml) was incubated with NENMBA (100 µM with Rb, 40
µM with Na) at 37 °C at pH 7.5 (40 mM Hepes)
for 2 min with the concentrations of cations indicated and activity was
assayed as described under ``Materials and
Methods.''
Figure 5:
Effect of varying pH on the inhibition of
purified Na,K-ATPase by NENMBA. The enzyme (0.05 mg/ml) was incubated
with NENMBA (100 µM) at 37 °C for 2 min, and activity
was assayed as described under ``Materials and Methods.'' The
buffers used were: Mes (6.5), Hepes (7.0, 7.5), Taps (8.0, 8.5), and
Ches (9.0).
Figure 6:
NENMBA access to inhibitory site. The
enzyme (0.05 mg/ml) was incubated with NENMBA (100 µM) at
37 °C for 2 min at pH 7.5 (40 mM Hepes), and then half the
sample was pelleted rapidly (450,000
Fig. 3shows that increasing [NENMBA] increases the
rate of inhibition of the Na pump. If fully inhibited enzyme (<2%
residual activity) is pelleted by centrifugation, washed in
inhibitor-free buffer, and resuspended in buffer, no recovery of enzyme
activity is observed. Thus, the dissociation of the inhibitor appears
to be an extremely slow process. The lack of recovery is independent of
the composition of the resuspension media (± Rb, ± ATP,
± BSA, ± Na). The presence of monovalent cations (Na, K,
or Rb) in the incubation medium can prevent the inhibitory binding of
NENMBA to the Na pump. These cations protect the enzyme with
concentrations in the mM range: K
Figure 7:
Photoincorporation of NENMBA into
Na,K-ATPase
In the present work we have shown that NENMBA, a
pyrazine-substituted amiloride derivative, is a potent inhibitor of the
Na,K-ATPase. The inhibition is prevented by the simultaneous presence
of monovalent cations, which are transported by the Na pump, and the
inhibitor is covalently coupled to the protein upon irradiation at 313
nm. It has been known for some time that amiloride itself inhibits
the Na pump, albeit with low affinity, and that pyrazine-ring
substituted amiloride derivatives exhibit an enhanced affinity for the
Na pump compared to the parent compound(34, 35) .
However, these studies were performed either on various cell lines or
on Na pump preparations which had 1.5% the specific activity of the
enzyme used in this report. The intent of the present studies was to
exploit the binding of photoactivatable amiloride derivatives
to the Na pump, so as to localize non-acidic amino acid residues in the
cation transport domain of the enzyme. The fundamental requirement for
the success of this strategy is that the cation photoaffinity probes
bind with high affinity to Na pump prior to photolysis, in a
cation-preventable manner, so that reasonably specific covalent
incorporation of the probe into the protein can subsequently be
achieved.
The tight binding or
``occlusion'' of both Na Fig. 6shows that when
partially inhibited Na pump is pelleted and resuspended in NENMBA-free
buffer, the observed rate of inhibition is reduced 2-3-fold.
Several conclusions can be drawn from this observation. First, as
observed previously and mentioned above, inhibition is not relieved by
resuspension in inhibitor-free medium, i.e. there is a very
slow off-rate from the inhibitory site. Second, it seems highly likely
that inhibitor in the membrane phase is associated with the slow
development of inhibition. NENMBA has been found to be highly
hydrophobic, having a partition coefficient of 200:1 between chloroform
and 0.1 M phosphate buffer at pH 7.4 (24) . We
estimate that the volume ratio of the pellet/resuspension medium is
about 1:100. We would expect the partition equilibrium to be
re-established very rapidly on resuspension, and thus the concentration
of NENMBA in the membrane following resuspension would be about
one-half of its value prior to resuspension, and the rate of
development of inhibition would fall by about 2-fold, which is what is
observed (Fig. 6). Therefore, the probe gains access to its
inhibitory site after it has partitioned into the lipid
environment. If NENMBA bound to the Na pump directly from the aqueous
solution, pelleting and resuspension of the partially inhibited protein
should effectively stop any further inhibition; such is not the case.
NENMBA has a pK
Second, NENMBA promises to be a useful
cation binding site photo-activated probe since the nitroanisole
chromophore reacts by displacement of the methoxy group through a
photoaromatic nucleophilic substitution reaction(43) . These
types of chromophores are stable upon irradiation in the absence of
nucleophiles(44) . This property makes them rather
different from the more widely used aromatic azides, which produce
reactive nitrenes upon illumination. If these nitrenes do not react
with the target protein, they decompose to inert side products; for
this reason, observed yields of covalent incorporation of such
photoaffinity probes are often very low. In principle, the efficiency
of covalent modification of the target enzyme can be much higher with
the chromophore used in NENMBA, since it returns to ground state
starting materials, if it does not encounter a nearby nucleophile, and
so can be re-excited repeatedly during irradiation until a productive
encounter occurs. Two other types of cation binding site chemical
probes have been used with the Na pump. The most extensively used are
carbodiimides, which react with carboxyls (i.e. glutamates and
aspartates) to produce an activated ester, which can react with a
nucleophile to produce an amide; thus, intramolecular cross-linking is
possible with these probes(16, 19) . This disadvantage
has been recently circumvented by use of a cationic substituted
diazomethane (DEAC), which also reacts chemo-selectively with
carboxylates to form stable ester derivatives of amino acids
directly(22, 23) . These reagents have been used
because it has been thought for some time that negatively charged amino
acid residues might be involved in cation binding and transport.
However, inspection of the structures of naturally occurring monovalent
cation ionophores suggests that serines, threonines, and tyrosines
could also be involved in cation co-ordination of Na and K by the Na
pump. Furthermore, the first atomic-resolution crystal structure of a
Na/K binding enzyme (the dialkylglycine decarboxylase) shows no Asp or Glu involved in Na/K binding at one of its two cation
co-ordination centers(45) . In summary, we found that four
photoactivatable amiloride analogues inhibit the Na,K-ATPase and
monovalent cations protect the enzyme only when the photolabile
substituent is on the pyrazine ring. Upon illumination, NENMBA is
covalently incorporated into the
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10353-10358
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
H]ADP (and hence ATP) binding. The rate of
inhibition increased with increasing concentrations of NENMBA.
Extensive washing of NENMBA-inhibited enzyme did not restore ATPase
activity, showing that NENMBA has an extremely slow off-rate for
dissociation from its inhibitory site. Partially inhibited enzyme could
be rapidly pelleted and resuspended in NENMBA-free buffer and
inhibition was observed to continue, albeit at a somewhat diminished
rate, suggesting that NENMBA gains access to its inhibitory site after
partitioning into the lipid phase rather than directly from the aqueous
solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent
incorporation of the reagent into the 
-subunit of the Na,K-ATPase,
as observed by separation of labeled protein on a Laemmli gel and
Western analysis using a polyclonal amiloride antibody. Almost all of
the covalent labeling could be prevented by the presence of Rb in the
incubation and labeling medium. These results suggest that NENMBA
inhibits the Na,K-ATPase by disruption of the cation transport domain
rather than the catalytic domain of the enzyme and that it promises to
be a useful tool for cation site localization.
-subunit and a 
-subunit. The -subunit consists of about
1020 amino acid residues and has been cloned from several
sources(1, 2, 3, 4, 5, 6) .
The -subunit is composed of about 300 amino
acids(7, 8, 9) . The enzyme undergoes a
series of conformational changes through its catalytic cycle, which
couple binding of ATP and enzyme phosphorylation to the active
transport of the monovalent cations against their electrochemical
potential gradients (for reviews see (10, 11, 12) ). In its normal transport mode,
the enzyme is phosphorylated by ATP when cation binding residues which
selectively bind sodium are exposed to the cytosol; the enzyme then
undergoes a major conformational transition and transports three
sodiums across the cell membrane, where they dissociate to the external
medium. The (phospho-) enzyme cation ligating residues are now exposed
to the external medium and are more potassium-selective; two potassiums
(or cogeners for potassium such as rubidium, cesium, etc.) bind, and
the phospho-enzyme bond is hydrolyzed. The potassiums are then occluded
within the protein, and the enzyme undergoes the second major
conformational transition of its catalytic cycle when ATP binds with
low affinity (but does not phosphorylate the protein) and so
facilitates the release of potassium to the cytosol. The binding of
sodium ions then reinitiates the cycle.
)a stable diazomethane analogue, which reacts
specifically and unambiguously with carboxylates to form stable ester
derivatives of the protein, has been recently shown to modify Glu-779
in a cation-preventable manner(22, 23) . However, by
analogy with the naturally occurring ionophores, we would infer that
not only glutamate or aspartate side chains are involved but the
electron lone pairs of other, neutral residues might also be involved
in monovalent cation transport.-subunit of the Na pump upon irradiation, as shown by Western
analysis using a polyclonal antibody. A preliminary report of some of
these results has appeared(26) .
Materials
Amiloride derivatives were a gift from
Merck, Sharp and Dohme, West Point, PA. ATP, ADP, imidazole, Hepes,
Mes, Taps, Ches, Tris, and bovine serum albumin (BSA), dithiothreitol,
sucrose, ultrapure urea, and Tricine were from Sigma; L-1-tosylamide-2-phenylethylchloromethyl ketone-treated
trypsin was from Worthington Corporation. -Mercaptoethanol, sodium
dodecyl sulfate, ammonium persulfate, Coomassie R-250, and low
molecular weight standards were from Bio-Rad. Acrylamide and
bisacrylamide were obtained through Boehringer Mannheim. Nitrocellulose
was from Millipore.Enzyme Isolation and Assays
Na,K-ATPase was
purified from dog kidneys according to Jørgensen (27) with the modification of Liang and Winter(28) .
The enzyme was greater than 95% pure as judged by SDS-PAGE (see Fig. 9
in (33) ). The standard assay medium for Na,K-ATPase activity
was (mM): EDTA, 0.5; NaCl, 130; KCl, 30; MgCl
, 3;
ATP, 3: imidazole, 50; pH 7.2 (25 °C); and about 0.8 µg/ml
enzyme protein. Unless specified, it also contained 0.3 mg/ml BSA. The
suspension was incubated at 37 °C for 15 min and then the P
released determined as reported by Brotherus et
al.(29) . The Na,K-ATPase activity was the difference
between the ATP hydrolysis measured in the presence and absence of 0.5
mM ouabain. The enzyme used in these studies had a specific
activity of 20-26 µmol of P released
mg
min, a negligible
ouabain-insensitive ATP hydrolysis and maximal phosphorylation by
P at 37 °C in the presence of ouabain of about
2.5-3.0 nmol mg protein. Protein was
determined by the method of Lowry et al.(30) using
BSA as a standard.Treatment with Amiloride Derivatives
Incubation of
purified Na,K-ATPase with each reagent (100 µM) was
performed at 37 °C by incubating the enzyme (0.05 µg/ml) in a
medium containing 50 mM Hepes (except where noted) with 2
mM EDTA. The reagents were initially dissolved in
MeSO so that the final concentration of MeSO in
the incubation medium was 2-10%. The reactions were stopped by a
60-fold dilution into ice-cold assay medium. The results presented in Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6are
representative of at least three experiments in which all samples were
run in duplicate. The range between duplicates was less than 10%.
ATPase activity did not vary between experiments by more than 10% about
the mean value shown in Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6.
), 40 µM (
), 60 µM (
), 80 µM (
), and 100 µM (
) NENMBA at 37 °C at
pH 7.5 (40 mM Hepes), and aliquots were assayed at the time
points indicated.
g, 4 °C, 2
min) and resuspended in NENMBA-free buffer. Aliquots were taken at the
time points indicated, and activity was assayed as described under
``Materials and Methods.''
Irradiation Procedure
Preparative photolabeling of
Na pump protein with NENMBA was performed by incubation of the enzyme
(1 mg/ml) with NENMBA (1 mM) for 10 min at 37 °C. The
enzyme was then pelleted, washed, and resuspended in NENMBA-free Hepes
(50 mM) and EDTA (2 mM). 200-µl aliquots were
irradiated in a 1-mm pathlength cuvette for 2 min with a 1000-watt high
pressure Hg arc lamp filtered through a 313 ± 10 nm band-pass
filter (Oriel) in order to excite specifically the nitroaromatic
chomophore. Gel electrophoresis was carried out on intact Na pump using
a linear (10%) SDS-PAGE system after Laemmli (31) .Immunoblots
Serum containing polyclonal antibodies
raised against amiloride conjugated to keyhole limpet hemocyanin via
the 5-position of the pyrazine ring (32) were purified by twice
treating 25 ml of a 1:100 dilution (81 µg) in TBS (200 mM NaCl, 50 mM Tris, pH 7.5) with 100 µg of purified Na
pump spotted onto nitrocellulose (and blocked with milk), so removing
any cross-reactivity from the serum. After electrophoresis of labeled
protein samples and protein transfer to nitrocellulose by
electroblotting, the blot was incubated at room temperature for 1 h
with 5% milk protein and then incubated with a 1:100,000 dilution of
the primary antibody overnight at 4 °C. The blot was washed five
times with TBS and then incubated at room temperature for 2 h with a
1:2000 dilution alkaline phosphatase goat anti-rabbit antibodies in
buffer, followed by washing five times in TBS. Bound antibodies were
then detected with Sigma Fast.High Affinity ADP Binding
Nucleotide binding was
measured as described previously (33) in a medium containing
(mM): Hepes/imidazole, 30; pH (25 °C) 7.2; NaEDTA, 0.1;
NaCl, 5; [H]ADP, 0.10; and 0.5 mg/ml protein. The
suspension was shaken at 4 °C for 30 s and centrifuged at 400,000
g for 10 min. The pellet was resuspended in 2.5 M NaOH, and radioactivity and protein concentration were determined.
Radioactivity bound to the enzyme in the presence of 0.5 mM ATP was subtracted from the experimental values as a correction
for nonspecific (or low affinity) binding.
Characteristics of Enzyme Inhibition
Incubation
of purified Na,K-ATPase with the four amiloride derivatives shown in Fig. 1resulted in inhibition of the enzyme activity to varying
degrees (Fig. 2). The two ``channel-specific''
compounds (NMBA and bromobenzomil) inhibited the enzyme
(
30-40% under the conditions shown) but the presence of Rb
ions in the incubation medium were without effect. Under the same
conditions, the ``exchanger-specific'' compounds (NENMBA and
6-bromo-5(N,N-cyclohexyl)amiloride) produced
70-90% inhibition of Na,K-ATPase and cation prevention of
inhibition was observed. In the case of NENMBA, the protection against
inhibition was almost complete. It should be emphasized at this point
that even though all these probes can potentially photolabel the Na
pump upon illumination (300-360 nm), the inhibition of the Na
pump as illustrated in Fig. 2is occurring in the absence of
illumination. The most effective prevention of inhibition by Rb was
observed with NENMBA; thus, this probe was examined in more detail. 
for
Rb (which is recognized as a cogener for K by the Na pump) is about 1
mM (see Fig. 4A), and for Na is about 7 mM (Fig. 4B), when a concentration of 100 µM NENMBA was used to inhibit the enzyme at 37 °C for 10 min.
However, since NENMBA has an extremely slow off-rate, eventually the
enzyme is fully inhibited even in the presence of 100 mM monovalent cations.Effect of pH
When the pH of the medium is varied,
the rate of binding of NENMBA to its inhibitory site on the Na pump
changes. As the pH is increased from 6.5 to 9.0, the rate of
development of inhibition increases (Fig. 5). The effect of pH
on the inhibitor:protein interaction is probably more complex than
simple titration of the inhibitor, the pK
of which
is 8.3 (24) . We believe that partitioning into the lipid phase
occurs prior to inhibition (see below) and the partition coefficient is
likely to increase with increasing pH.Access Pathway to Inhibitory Site
The slow
development of inhibition and its very slow (if any) reversibility,
together with the greater effectiveness of the unprotonated reagent,
raised the question as to whether NENMBA bound directly to the
inhibitory site from the solution or first partitioned into the
membrane phase. Experiments were designed to examine these
possibilities. A sample of enzyme was incubated with NENMBA at 37
°C and when about 50% inhibition had been attained, half the enzyme
was pelleted at 450,000 g at 4 °C for 5 min and
resuspended in inhibitor-free buffer at 37 °C, and the incubation
was continued. As shown in Fig. 6, a slower rate of inhibition
is seen in the resuspended enzyme than with the uninterrupted
incubation.High Affinity Nucleotide Binding
Although the
greatest protection against inhibition was shown by cations and only
little (if any) protection by ATP (see Fig. 2), it was important
to see whether or not the ATP binding domain was greatly affected by
the presence of the inhibitor. This experiment was possible because of
the extremely slow reversibility of the inhibition due to NENMBA
addition. It was found that both native and NENMBA-treated enzyme could
bind [H]ADP to the same extent; about 2.5 nmol of
ADP was specifically bound per milligram of enzyme. This level of
binding is very similar to that previously observed for native enzyme (33) and DEAC-treated enzyme(22) .Photoincorporation and Location of
Labeling
Irradiation of fully inhibited enzyme at 313 ±
10 nm with the output from a high pressure Hg arc lamp (only exciting
the nitroaromatic chromophore; (25) ) results in the covalent
incorporation of the probe specifically into the -subunit of the
Na pump (see Fig. 7). Western analysis of Na pump, after
separation by SDS-PAGE and transfer to nitrocellulose, using an
amiloride polyclonal antibody, shows that irradiation is necessary for
covalent incorporation (Fig. 7, lanes 3 and 4;
there is no cross-reactivity of the antibody with the Na pump, lane
2). Irradiation of Na pump in the presence of NENMBA and Rb shows
that there is very little, if any, nonspecific photoincorporation of
the probe into the protein (lane 5). Staining of the
nitrocellulose membrane after transfer but prior to blocking with milk
with the reversible dye, Ponceau Red, showed that approximately the
same amount of protein had been transferred in each lane of Fig. 7(not shown).
-subunit. The enzyme (0.05 mg/ml) was incubated with
NENMBA (100 µM) at 37 °C for 5 min ± Rb (20
mM) at pH 7.5 (40 mM Hepes) and irradiated with a
high pressure Hg arc lamp for 2 min through a 313 nm narrow band-pass
filter, separated using a Laemmli gel (10%), and transferred to
nitrocellulose. Covalent incorporation of NENMBA was tested using a
polyclonal amiloride antibody. No signal was observed in the Western
analysis from NENMBA (lane 1), Na pump (lane 2), Na
pump and NENMBA without irradiation (lane 4). Irradiation
produces a stable chemical bond between NENMBA and Na pump
-subunit (lane 3); the presence of Rb (lane 5)
prevents most of this incorporation.
Structure-Activity Relationships
We examined the effects
of four amiloride derivatives, all of which are potentially
photoactivatable, on the ability of purified renal Na,K-ATPase to
hydrolyze ATP. All four compounds inhibited ATPase activity (Fig. 2). However, we found that it was only the
``exchanger-specific'' probes, NENMBA and
6-bromo-5(N,N-cyclohexyl)amiloride, which could be
prevented from binding to and inhibiting the Na pump by the presence of
monovalent cation ligands. It seems from this pharmacological profile
that the Na pump interacts with amiloride derivatives in a manner which
is more like other transport proteins such as the Na/Ca and Na/H
exchangers than epithelial Na channels. The Na/Ca and Na/H exchangers
are like the Na pump in that they undergo a series of conformational
transitions during their transport cycle in which the cation binding
sites are alternately exposed to each side of the membrane. However,
there is no homology in the primary structures or evidence of
similarity of cation binding site structure between these exchangers
and the Na,K-ATPase. Tight binding of one amiloride derivative
(phenamil) has also been previously observed on epithelial Na
channels(36) . David et al.(37) have recently
surveyed 15 amiloride derivatives (including NMBA) for their inhibitory
activity against the Na pump. They observed that pyrazine-substituted
amilorides were effective competitive inhibitors of Rb occlusion, with
concentrations in the tens of micromolar range, whereas
guanidine-substituted amilorides only seemed to prevent Rb occlusion at
very high concentrations. The rates of development or reversal of
inhibition were not reported, as the assay used in their study was
performed by incubating purified Na pump at room temperature for 3 min
with 
Rb in the absence and presence of varying
concentrations of amiloride derivatives.Ligand Prevention of NENMBA Inhibition
Since
cation prevention of inhibition of the Na,K-ATPase was clearest in the
case of NENMBA (Fig. 2), it was decided to examine these effects
in more detail (Fig. 4). Both Na
and
Rb (as a cogener for K), protected
the enzyme against inhibition of NENMBA. We have found that almost all
(90%) the inhibition by NENMBA can be prevented when 10 mM K
or Rb is included in the incubation medium (Fig. 4A),
and that Rb protects the enzyme with a K in the
1-2 mM range. Sodium does not prevent binding quite so
effectively; nevertheless, about 60-70% of the enzyme activity
can be preserved when about 60 mM Na is included in the
incubation medium. The K for this protective
effect is about 10 mM (Fig. 4B). Protection
against covalent inhibition by DEAC, a cation-site directed reagent,
has also been reported to be more effective and complete with K than
Na(22) . However, these protective effects of ligand cations
can be overcome simply by prolonging the incubation period. Once the
probe is bound to its inhibitory site, it exhibits an extremely slow
dissociation rate from this complex, as extensive washing of the enzyme
with ligands (Na,K-ATP) does not restore ATPase activity. Furthermore,
native and NENMBA-inhibited Na pump exhibited the same level of high
affinity nucleotide binding, suggesting that NENMBA does not perturb
greatly the catalytic domain of the enzyme. This is encouraging for the
use of NENMBA as a cation-site probe, since it is desirable that such a
reagent should have effects which, as far as possible, are limited to
the cation-binding domain. and K have been well characterized kinetically and it is thought that
these intermediates do in fact form part of the normal catalytic cycle
of the Na pump(10, 38, 39, 40) . An
important characteristic of the cation occlusion phenomenon is the slow
association and dissociation of the cations. It is tempting to
speculate that since NENMBA does not seem to dissociate from the enzyme
it also becomes occluded by the Na pump; however, since other
substituted amines(41) , including guanidines(42) , do
not seem to be occluded by the Na,K-ATPase, we deem it unlikely that
NENMBA is exceptional in this regard. of about 8.3(24) ; thus,
as the pH is raised from 6.5 to 9.0 (Fig. 5), the reagent
deprotonates and partitions more completely into the membrane. Entry to
the inhibitory site seems to occur from the membrane phase, so that the
rate of inhibition thus increases as the pH is raised.Localization of Labeling
One of the potential
disadvantages of photoaffinity labeling in general is the possibility
of producing nonspecific photo-incorporation of a probe, as irradiation
normally produces a hyper-reactive chemical species (nitrene or
carbene), which can even insert into C-H bonds. NENMBA is an attractive
chemical probe for monovalent cation binding residues in the
Na,K-ATPase for at least two reasons. First, the tight binding and slow
dissociation of NENMBA from the Na pump enabled fully inhibited enzyme
to be pelleted, washed, and resuspended in NENMBA-free buffer and
irradiated to produce cation-preventable, specific photolabeling of the
Na pump -subunit (Fig. 7, compare lanes 3 and 5). Radioactive NENMBA was not available; therefore, covalent
incorporation was probed by Western analysis of labeled Na pump
transferred onto nitrocellulose, and probed using the amiloride
polyclonal antibody. Even though NENMBA binding can completely prevent
ATP hydrolysis by the Na pump, this inhibition does not result in a
positive signal in the Western blot (lane 4), i.e..
irradiation is necessary for covalent incorporation of the
probe into the -subunit.-subunit as shown by Western
analysis using an amiloride specific antibody. Further studies are
under way to localize the amino acid residue labeled by NENMBA.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
