(Received for publication, June 28, 1995; and in revised form, September 28, 1995)
From the
When the gastric H,K
-ATPase
was solubilized by n-dodecyl
-D-maltoside and
electrophoresed in blue native-polyacrylamide gels (BN-PAGE), one major
band at about 360 kDa was observed. Since this band was recognized by
both monoclonal antibodies 1218 (anti-
) and wheat germ agglutinin
(anti-
), the H
,K
-ATPase in its
native state exists in a dimeric (
)
form. The
site of interaction between the heterodimers was determined using
Cu
-phenanthroline cross-linking. The
Cu
-phenanthroline reagent reacted with the
H
,K
-ATPase to produce an
,
-dimer and inhibited
H
,K
-ATPase activity. This
cross-linking and enzyme inhibition were prevented by ATP.
Cross-linking followed by N-ethylmaleimide blockade of
maleimide-reactive SH groups, then reduction and fluorescein
5-maleimide labeling, defined a single fluorescent tryptic peptide of
about 6.5 kDa that had been cross-linked. Since its N-terminal amino
acid is Val
, the peptide probably ends at Arg
or Arg
and Cys
and/or Cys
are probably within the region of closest contact between the two
-subunits.
The gastric H,K
-ATPase is a
member of the ion-motive, phosphorylating P-type ATPases which include,
in mammals, the Na
,K
,
Ca
, and
H
,K
-ATPases. This gastric enzyme
catalyzes an electroneutral exchange of extracytoplasmic K
for cytoplasmic H
O
(1) . The
gastric H
,K
-ATPase consists of two
subunits, a catalytic
-subunit and a heavily glycosylated
-subunit(2) . The catalytic
-subunit, a 114-kDa
protein(3, 4, 5) , contains the sites of
nucleotide binding and phosphorylation (6) and runs at a
relative molecular mass of 94-100 kDa in reducing SDS-PAGE. (
)The
-subunit(7, 8) , which has a
relative molecular mass of about 60-80 kDa in SDS-PAGE, has a
core protein of 34 kDa(9) . Using lectin-affinity
chromatography, the solubilized
H
,K
-ATPase
-subunit co-purified
with the
-subunit, and the luminal loop between the
membrane-spanning segments TM7 and TM8 of the
H
,K
-ATPase
-subunit was shown to
be a site of strong association with the
-subunit(10, 11, 12) .
The radiation
inactivation target size of the gastric
H,K
-ATPase was 444 ± 10 kDa in
the absence and 371 ± 39 kDa in the presence of
Mg
(15) , whereas H
transport
in reconstituted proteoliposomes was shown to have a mean target size
of 388 ± 48 kDa(13, 14, 15) .
Two-dimensional crystallization of membrane-bound
H
,K
-ATPase showed that the unit cell
dimension was 115.1 Å with p4 projection symmetry, suggesting
strong protein-protein interaction between (
)
protomers(16) . These studies suggest the
H
,K
-ATPase is likely to exist as a
dimeric (
)
-heterodimer in the original membrane.
To obtain specific information about the organization of the ATPase,
blue native (BN)-gel electrophoresis was used to identify its
oligomeric nature. Disulfide cross-linking using
Cu-phenanthroline was followed by identification of
the regions of cross-linking by fluorescein maleimide (F-MI) labeling
following NEM protection of maleimide accessible cysteines.
A 70-µl
sample of this solution was placed on top of a 6% (34:1
acrylamide/methylene bisacrylamide) to 13% (34:1 acrylamide/methylene
bisacrylamide) 1.5-mm thick gradient slab gel, using BN-polyacrylamide
gel containing 0.01% n-dodecyl -D-maltoside,
prepared by the method of Schägger and von
Jagow(19) . The cathode buffer consisted of 50 mM Tricine, 15 mM Bis-Tris, pH 7.0, and 0.02% Coomassie Blue
G-250, and the anode buffer consisted of 50 mM Bis-Tris/HCl,
pH 7.0. The gel was run for 2 h at 100 V constant voltage and then at
200 V constant voltage for 6 h. Two lanes were run for standard
molecular mass markers (one lane for jack bean urease to give 545- and
272-kDa molecular mass markers, the other lane for bovine serum albumin
giving 132- and 66-kDa molecular mass markers). The gel was destained
in a solution composed of 30% methanol and 10% acetic acid. Prior to
transfer to PVDF, the gel was washed for 2 h in a buffer composed of
0.1 M boric acid/NaOH, pH 8.8, 0.12% SDS with several changes
of buffer. Proteins of this Coomassie Blue-stained gel were
transblotted to a PVDF membrane using a solution of 10% methanol, 180
mM glycine, 25 mM Tris buffer.
The vesicular
H,K
-ATPase was resuspended in 50
mM Tris/HCl, pH 7.4, at a protein concentration of 1 mg/ml. A
200-µl aliquot of the gastric
H
,K
-ATPase was treated with 2 µl
of 10 mM Cu-phenanthroline complex and kept for 30 min on ice.
The reaction was stopped by addition of 2 µl of 0.5 M NEM
and 1 µl of 0.2 M EDTA on ice. The suspension was
centrifuged at 110,000
g (4 °C) for 45 min to
remove excess Cu-phenanthroline complex and other reagents. The pellet
was resuspended in 50 mM Tris/HCl, pH 7.0, at a protein
concentration of 1 mg/ml. The effects of ligands on the reaction were
examined by addition of the cross-linking reagent to enzyme solution
containing individually 2 mM ATP, ADP, Mg
,
200 mM KCl, 10 mM EDTA, or 1 mM HVO
.
The gastric
H,K
-ATPase was also solubilized with
Nonidet P-40 as described previously (10, 12) and
cross-linked. 1 mg of the enzyme was solubilized with 0.5 ml of 0.2%
Nonidet P-40, 50 mM Tris/HCl, pH 7.4, and centrifuged at
110,000
g for 15 min. The supernatant was reacted with
0.1 mM Cu-phenanthroline for 30 min on ice. Reaction with
Cu-phenanthroline was stopped by adding 10 mM NEM. The
reaction mixture was run on 5% Tricine gel after adding 40% sample
buffer.
Alternatively the proteins after reaction
with the Cu-phenanthroline complex were dissolved in 0.2% SDS, 1 mM NEM, 50 mM Tris/HCl, pH 7.0, at a protein concentration
of 1 mg/ml, and combined with a 30% volume of sample buffer (0.3 M Tris/HCl, pH 7.4, 10% SDS, 50% sucrose, and 0.025% bromphenol
blue). The samples were loaded on top of a 5-15% gradient SDS-gel
prepared as described above and run at 45 mA constant until the dye
reached the bottom of the gel. We obtained a migration of the proteins
such as the ,
-dimer at about 190 kDa, the
-subunit at
about 95 kDa, and the
-subunit at about 60-95 kDa in the
5-15% gradient SDS gel. For clearer separation of the
,
-dimer from monomer, we used a 5% Tricine gel. In this 5%
homogeneous Tricine gel, we observed that the
-subunit migrates
more slowly than the
-subunit, while the dimer migrates at 190 kDa
as expected.
The gels were stained by 0.1% Coomassie Blue in 45% methanol and 10% acetic acid or electrotransferred to PVDF membranes as described previously(12) .
Figure 1:
BN-PAGE of soluble
H,K
-ATPase. The gastric
H
,K
-ATPase was incubated in a buffer
composed of 50 mM Tris/HCl, pH 7.0, and 1% n-dodecyl
-D-maltoside, and solubilized as described under
``Experimental Procedures.'' Panel A shows the
Coomassie-stained PVDF membrane following BN-PAGE of the
H
,K
-ATPase and protein transblotting
from gel to PVDF, and Panel B shows Western blotting of mAb
1218 (lane 3) and WGA (lane 4). Lane 1 contained the molecular mass standards (jack bean urease; 545
(hexamer) and 272 kDa (trimer)). Lane 2 represents the
H
,K
-ATPase.
The Cu-phenanthroline
reaction with the membrane-bound gastric
H,K
-ATPase provided a 190 kDa-band
with an additional minor band near the top of gel, which was also
recognized by mAb 1218. The yield of the 190-kDa band was about 30%
based on optical scanning of the Coomassie Blue stain. Fig. 2illustrates the Coomassie Blue-stained gel (Panel
A) and the Western immunoblot using mAb 1218 and WGA (Panel
B). As shown in this figure, the band at 190 kDa (Fig. 2, Panel A, lanes 2, 3, and 4)
contains only the
-subunit, recognized by mAb 1218 (Panel
B, lane 2a). The
-subunit was shown to be same in
unreacted enzyme and Cu-phenanthroline-reacted enzyme, which indicates
that the
-subunit had not reacted with Cu-phenanthroline (Panel B, lanes 1b and 2b). When enzyme was
reacted with Cu-phenanthroline in the presence of ATP or Mg-ATP, the
,
-dimer was not formed (Fig. 2, lanes 5 and 6). Vanadate was also relatively effective in inhibiting
dimerization (data not shown). Other ligands such as K
and Mg
did not inhibit the formation of dimers (Fig. 2, lanes 3 and 4). When the
cross-linking reaction was carried out in the presence of chelating
agents such as EDTA, the formation of the
,
-dimer was
inhibited as shown in Fig. 2, lane 7. These chelating
agents are able to remove cupric ion from Cu-phenanthroline and prevent
Cu
complex formation (28) .
Figure 2:
SDS-PAGE of the membrane-bound
H,K
-ATPase treated with
Cu
-phenanthroline complex. Cu-phenanthroline reaction
with the H
,K
-ATPase and 5-15%
Tricine gradient gel electrophoresis of reacted
H
,K
-ATPase was carried out as
described under ``Experimental Procedures.'' Panel A shows the Coomassie-stained gel and Panel B represents
mAb 1218 staining (lanes 1a and 2a) and WGA staining (lanes 1b and 2b). Lane 1 represents
unreacted H
,K
-ATPase. Lane 2 represents an electrophoretic pattern of enzyme reacted with 0.1
mM Cu-phenanthroline. Lanes 3, 4, 5, 6, and 7 represent Cu-phenanthroline (0.1
mM) reaction with enzyme pretreated with 0.2 M KCl, 2
mM MgCl
, 2 mM ATP, 2 mM Mg-ATP,
and 10 mM EDTA, respectively. Lane 8 represents the
DTT reduced product of the reacted enzyme that is shown in lane
2.
As now shown
in Fig. 2, lane 8, the ,
-dimer induced by
Cu-phenanthroline reaction was reduced by DTT, showing that
cross-linking was due to disulfide formation and/or Cu-protein complex (28) .
Higher concentrations of Cu-phenanthroline (over 1 mM) resulted in a high level of protein aggregation on the top of gel, showing a loss of selectivity in the cross-linking reaction at higher concentrations of the cross-linking reagent. This result determined the level of cross-linking reagent used in these studies.
Figure 3:
Inhibition of ATPase activity by
Cu-phenanthroline. The gastric
H,K
-ATPase was reacted with
Cu-phenanthroline (0.1-100 µM) and centrifuged to
remove unreacted Cu-phenanthroline. ATPase activity of the washed
enzyme was measured as described under ``Experimental
Procedures.'' Panel A shows that the inhibition of the
gastric H
,K
-ATPase is dependent on
the concentration of Cu-phenanthroline. Panel B shows that ATP
pretreated enzyme was not affected by
Cu-phenanthroline.
When the
H,K
-ATPase that had been treated with
Cu-phenanthroline was exposed to either 0.1 M DTT or 0.2 M
-mercaptoethanol, the
,
-dimer was reduced to
provide the monomer as shown in Fig. 2, lane 8.
Treatment with DTT alone inhibited enzyme activity by about 80% and
treatment of the cross-linked enzyme restored 20% of control activity
showing probable reversal of the inhibition induced by cross-linking
(data not shown).
Figure 4:
Trypsin digestion of the
H,K
-ATPase labeled by F-MI. The
Cu-phenanthroline reacted H
,K
-ATPase
was treated with excess NEM for blocking free thiols. The NEM-treated
protein was reduced by DTT and labeled by F-MI as described under
``Experimental Procedure.'' Lane 1 shows
F-MI-labeled product, and lane 2 shows membrane-bound tryptic
digest. Lane 4 shows cytoplasmic tryptic fragments presented
in the supernatant. The F-MI-labeled tryptic fragment had the
N-terminal sequence of VLGFXQLYL. Lane 3 contains the
molecular mass standards, i.e. 106, 80, 47, 31, 20, 17, 16,
14.5, 10.6, 8.2, and 6.2 kDa.
From the BN-PAGE study, we found that the oligomeric form of
the solubilized H,K
-ATPase to be an
(
)
-dimer. This (
)
-dimer was
found even after the treatment with ligands such as ATP, ADP, ATP-CDTA,
and Mg-ATP. The size of the dimeric (
)
form on
the BN-PAGE was about 360 kDa. In this BN-PAGE, measuring molecular
mass is not as accurate as on SDS-PAGE given that the binding capacity
of Coomassie Blue G-250 differs between proteins(29) . However,
the presence of the band at about 360 kDa indicates that the dimeric
(
)
complex migrates at the expected molecular
mass for such a dimer. This size is close to that obtained by radiation
inactivation of either ATPase activity in the presence of
Mg
, i.e. 371 ± 39 kDa (15) or
H
-transport by the enzyme which had provided indirect
evidence for a dimeric form of the enzyme(13) .
Two-dimensional crystals of the gastric
H,K
-ATPase induced by an imidazole
buffer containing Mg
and vanadate showed a single
tetragonal shape with p4 projection symmetry(16) . The
asymmetric unit was thought to consist of one pear-shaped protein
domain corresponding to a H
,K
-ATPase
protomer. From the projection map, the contact regions between adjacent
protomers were relatively protein dense, indicating protein-protein
interaction and the possible presence of tetramers. We found one minor
band at about 720 kDa on BN-PAGE gel. This molecular size suggests the
presence of a tetrameric (
)
form but this form is
minor compared to the (
)
form.
When the
cross-linked enzyme was solubilized and run on SDS gels, no evidence
was found for any -subunit association suggesting that the
(
)
-dimer results from
-
association.
Since Cu-phenanthroline reaction occurs via
Cu
-cysteine complex formation(28) , one or
more cysteines of the
-subunits must have very close proximity to
each other. These data provide direct evidence for a dimeric or higher
order oligomer of the gastric
H
,K
-ATPase.
Cu-phenanthroline
reaction with the H,K
-ATPase resulted
in inhibition of the H
,K
-ATPase
activity and formation of a stable
,
-dimer. Dimer formation
was not affected by K
or Mg
but was
prevented by ATP even in the presence of CDTA and partially by
HVO
. These data suggest that ATP binding
may alter the distance between the cysteines via conformational changes
or mask the cysteines available for Cu-phenanthroline reaction.
Cu-phenanthroline also produces ,
-dimers with the
Na
,K
-ATPase(23, 24, 25, 26) .
In contrast to the gastric ATPase, in this closely homologous enzyme
dimer formation was induced by the addition of ATP (24) and ADP
and K
inhibited
,
-dimer
formation(25) .
The C-terminal 64-kDa peptide of the
Na,K
-ATPase had been shown to be
essential for dimerization(26) . Recently, using various
chimeras, a cytoplasmic region (no. 554-785) in the
-subunit
of the Na
,K
-ATPase was reported to be
necessary for specific
-
association(30) . When the
site of cross-linking in the
H
,K
-ATPase was identified as detailed
above, it was found to be within a 6.5-kDa fragment beginning at
Val
and ending at Arg
or Arg
,
based on molecular size. This suggests that Cys
and/or
Cys
are the cross-linked residues in the gastric ATPase.
The N-terminal 55 amino acid sequence of this domain in the
Na,K
-ATPase (no. 554-609) is
very similar to the fragment of the
H
,K
-ATPase defined biochemically as
the major site of cross-linking in this study. In particular, when the
sequences of the H
,K
-ATPase around
Cys
and Cys
are compared with the sequences
of the Na
,K
-ATPase around Cys
and Cys
, very high homology is seen. Thus, the
sequence, VLGFC, containing the first of these cysteines is identical
in both the H
,K
- and
Na
,K
-ATPase and the C-terminal
25-amino acid sequence of the
H
,K
-ATPase tryptic fragment (no.
592-617) containing the second of these cysteines shows very high
homology with the corresponding region (no. 583-607) of the
Na
,K
-ATPase. The high homology of
these regions suggests that the Cu-phenanthroline dependent
dimerization probably occurs in this domain of both enzymes via
cross-linking of the corresponding cysteines. This region of close
association is within the nucleotide binding domain of the ATPases
which is consistent with the effects of nucleotides on cross-linking.
Other regions of the enzyme could also be in close contact, but not
revealed by this particular reagent.