(Received for publication, June 25, 1995; and in revised form, August 21, 1995)
From the
To identify interfaces of - and
-subunits of
Na
/K
-ATPase, and contact points
between different regions of the same
-subunit, purified kidney
enzyme preparations whose
-subunits were subjected to controlled
proteolysis in different ways were solubilized with digitonin to
disrupt intersubunit
,
-interactions, and oxidatively
cross-linked. The following disulfide cross-linked products were
identified by gel electrophoresis, staining with specific antibodies,
and N-terminal analysis. 1) In the enzyme that was partially cleaved at
Arg
-Ala
, the cross-linked products were an
,
-dimer, a dimer of N-terminal and C-terminal
fragments, and a trimer of
and the two
fragments. 2) From
an extensively digested enzyme that contained the 22-kDa C-terminal and
several smaller fragments of
, two cross-linked products were
obtained. One was a dimer of the 22-kDa C-terminal peptide and an
11-kDa N-terminal peptide containing the first two intramembrane
helices of
(H
-H
). The other was a trimer
of
, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked
products of a preparation partially cleaved at
Leu
-Ala
were an
,
-dimer and a
dimer of
and the 83-kDa C-terminal fragment.
Assuming the most
likely 10-span model of , these findings indicate that (a) the single intramembrane helix of
is in contact with
portions of H
-H
intramembrane helices of
; and (b) there is close contact between N-terminal
H
-H
and C-terminal H
-H
segments of
; with the most probable interacting helices being the
H
,H
-pair and the
H
,H
-pair.
Na/K
-ATPase is the intrinsic
enzyme of the plasma membrane that carries out the coupled active
transport of Na
and K
in most
eucaryotic cells. The enzyme consists of two subunits,
and
,
that are associated noncovalently in molar stoichiometry of
1:1(1, 2) . The
-subunit, which is often called
the catalytic subunit, is phosphorylated and dephosphorylated in the
course of ATP hydrolysis, contains a number of residues that have been
implicated in nucleotide, ouabain, and cation bindings to the enzyme,
and exhibits several characteristic patterns of proteolytic digestion
when the enzyme is in different ligand-induced conformational
states(3, 4) . It has been known for a long time,
however, that both subunits are essential to normal
function(5, 6) , and that the ligand-induced
conformational transitions are transmitted across the
,
-interface(7, 8, 9) . More recent
studies have also indicated the role of the
-subunit in the
regulation of K
binding to the
enzyme(10, 11, 12) , and have provided
further evidence for the involvement of both subunits in the
enzyme's conformational transitions(13, 14) . It
is evident, therefore, that identification of the interfaces between
the two subunits is necessary for the eventual definition of the
structure-function relationship of the enzyme. Since high resolution
structures are not available, other approaches must be used for such
structural studies. Locations of some
,
-interfaces have been
surmised from experiments on the structural requirements for the
assembly of the
,
-complex(11, 15, 16, 17, 18, 19) ,
and from molecular modeling
studies(20, 21, 22) . In the work presented
here, we have used controlled proteolysis and chemical cross-linking
techniques to identify some of the contact domains of the two subunits
of the purified canine kidney enzyme. Detergent-solubilized
preparations were utilized for these studies, because solubilization
prevents intersubunit
,
-interactions, but allows continued
,
-associations(23) . The results have also provided
new information regarding the relative orientations of the
intramembrane segments of an
-subunit within the three-dimensional
structure of the enzyme. Preliminary accounts of portions of this work
have been presented(24, 25) .
Purified membrane-bound
Na/K
-ATPase of canine kidney medulla,
with specific activity in the range of 1000-1600 µmol of ATP
hydrolyzed/mg of protein/h, was prepared and assayed as described
before(26) . Such preparations were subjected to controlled
proteolysis by the following well established procedures. 1) The enzyme
was exposed to trypsin in the presence of K
as
described (27) to obtain a preparation in which about half of
the
-subunit was cleaved at Arg
-Ala
.
2) The enzyme was exposed to chymotrypsin in the presence of
Na
(28) to obtain a preparation with partial
cleavage of the
-subunit at Leu
-Ala
.
3) Extensive digestion of the enzyme in the presence of trypsin,
Rb
, and EDTA was performed at pH 8.5 according to
Karlish et al.(29) to remove large portions of the
cytoplasmic domains of the
-subunit, and obtain a preparation
containing the C-terminal 22-kDa fragment and several smaller fragments
of the
-subunit(29, 30) . In the three
preparations subjected to proteolysis as indicated above, the
-subunit is essentially intact.
Native or digested enzyme
preparations were solubilized by mixing a suspension of the enzyme (2
mg/ml) in 10 mM Tris-HCl (pH 7.4) with an equal volume of 6
mg/ml digitonin, stirring for 5 min at 24 °C, and centrifugation in
Beckman Airfuge for 30 min at 100,000 g. The clear
supernatant containing the solubilized enzyme was collected.
For
cross-linking, an aliquot of the above digitonin-solubilized enzyme was
mixed with an equal volume of a solution containing 0.25 mM CuSO, and 10 mM Tris-HCl (pH 7.4), and
incubated for 10 min at 24 °C. The reaction was terminated by
addition of SDS to a final concentration of 2%, and samples were
subjected to electrophoresis either on SDS-polyacrylamide gels (5 or
7.5%) at pH 2.4(27) , or on
Tricine(
)/SDS-polyacrylamide gels (31) using 10 or
16.5% gels. These gel systems have been used before for the resolution
of the three digested preparations used
here(10, 27, 28, 29, 30) .
Unless indicated otherwise, sulfhydryl reagents were omitted from all
buffers used for electrophoresis to preserve the cross-linked products.
For Western blot analysis, peptide bands were transferred to
nitrocellulose sheets, probed with specific antisera, and detected
using appropriate second antibodies conjugated to alkaline phosphatase (32) . The blots were either photographed or quantified by a
scanning densitometer (Bio-Rad). The scans of multiple lanes presented
in the same figure are quantitatively comparable. N-terminal analyses
of the peptide bands were done as described before(28) . For
the smaller peptides resolved on 16.5% Tricine/SDS gels, identical
bands from several gels were combined, electroeluted, electrophoresed
on a second 10% Tricine/SDS gel, and then transferred to polyvinylidine
difluoride membrane for sequencing.
Trypsin (Type III-S, bovine
pancreas), -chymotrypsin (type II, bovine pancreas), and
trypsin-chymotrypsin inhibitor (soybean) were obtained from Sigma.
Polyclonal antibodies specific for the sequences of sheep kidney
-subunit (33) were provided by Dr. W. J. Ball (University
of Cincinnati). These antibodies were against residues 16-30,
residues 111-122, and residues 1003-1013. As expected from
the comparison of sheep and canine sequences(34, 35) ,
the three antibodies reacted with the canine
-subunit on Western
blots. Two additional polyclonal antibodies were obtained by
conventional immunization of rabbits using denatured
- and
-subunits of canine kidney
Na
/K
-ATPase as antigens. These were
prepared by resolving sufficient quantities of the purified enzyme on
preparative SDS-polyacrylamide gels, and electroelution of the
separated subunits.
Previous studies of several laboratories have shown that when
detergent-solubilized preparations of
Na/K
-ATPase are subjected to
oxidative cross-linking catalyzed by Cu
or a complex
of Cu
and o-phenanthroline, the major
cross-linked product is the
,
-dimer(1, 2, 8, 23, 36, 37, 38) .
Experiments of Fig. 1A, lanes 1 and 2, show
the results of Cu
-induced cross-linking of the
digitonin-solubilized enzyme. In agreement with previous observations
on the digitonin-solubilized enzyme(1) , the quantities of
- and
-monomers decreased after cross-linking, a single
cross-linked product was formed, and the mobility of the remaining
was altered. That the cross-linked product is indeed an
,
-dimer is indicated not only by its mobility, but also by
its reactivities with a
-specific antibody (Fig. 1B,
lanes 1 and 2) and several
-specific antibodies (e.g.Fig. 4C, lanes 1 and 2). In the
remaining experiments presented below, enzyme preparations whose
-subunits were subjected to controlled proteolysis in three
different ways were similarly solubilized and cross-linked; and the
identifications of the cross-linked products were attempted.
Figure 1:
A,
Cu-induced cross-linking of the digitonin-solubilized
Na
/K
-ATPase with
cleavage at
Arg
-Ala
. Cleavage, cross-linking and
electrophoresis on 5% acid gels were done as described under
``Experimental Procedures.'' Lane 1, control enzyme
without cross-linking. Lane 2, cross-linked control enzyme. Lane 3, partially cleaved enzyme prior to cross-linking. Lane 4, partially cleaved enzyme after cross-linking.
Positions of prestained molecular mass markers are indicated by arrows on the right. B, reactivities of the
cross-linked products of the native
Na
/K
-ATPase and the enzyme cleaved at
Arg
-Ala
with a
-specific antibody.
Gels similar to those of A were blotted, stained, and scanned
as described under ``Experimental Procedures.'' Lane numbers
are the same as those of A.
Figure 4:
A,
cross-linking of solubilized
Na/K
-ATPase with
cleavage at
Leu
-Ala
. Cleavage, cross-linking, and
resolution on 5% gels was done as described under ``Experimental
Procedures.'' Lane 1, control enzyme before
cross-linking. Lane 2, control enzyme after cross-linking. Lane 3, partially cleaved enzyme before cross-linking. Lane 4, partially cleaved enzyme after cross-linking. B and C, reactivities of the cross-linked products of the
native Na
/K
-ATPase and the enzyme
cleaved at Leu
-Ala
with a
-specific
antibody (B) and an antibody against sequence 111-122 of
(C). Gels similar to those of A were blotted,
stained, and scanned. Lane numbers are the same as those used in A.
Figure 2:
A, cross-linking of the solubilized
Na/K
-ATPase whose
is
extensively digested. Digestion, cross-linking, and electrophoresis on
7.5% gels were done as described under ``Experimental
Procedures.'' Lane 1, control enzyme prior to
cross-linking. Lane 2, control enzyme after cross-linking. Lane 3, extensively digested enzyme before cross-linking. Lane 4, digested enzyme after cross-linking. Lane 5,
prestained molecular mass markers (kDa): 106, 80, 49.5, 32, 27.5, 18.5. B, reactivities of the cross-linked products of the native and
the extensively digested Na
/K
-ATPase
with a
-specific antibody. Gels similar to those of A were probed with the antibody. Lane 5 contained
prestained markers.
Since the gels used in the experiments
of Fig. 2were not suitable for the detection of smaller
peptides of the extensively digested enzyme, Tricine gels were also
used to resolve this preparation before and after cross-linking. Prior
to cross-linking, on the lower portions of these gels we noted two
prominent bands below the 22-kDa peptide (Fig. 3, lane
1). After cross-linking, intensities of the 22-kDa band and one of
the other bands decreased, while a product with mobility lower than
that of 22-kDa band was formed (Fig. 3, lane 2). The
product bands from a number of gels were combined, electrophoresed
again, and used for further identification. N-terminal analysis (Table 1) identified the 22-kDa C-terminal and the 11-kDa
N-terminal fragments, in 1:1 ratio, as the major components of the
product. As expected from the data of Table 1, the cross-linked
product reacted with antibodies against 1003-1013 and
111-122 sequences of on Western blots (not shown).
Figure 3:
Cross-linking of solubilized
Na/K
-ATPase whose
is
extensively digested, and its resolution on a Tricine gel. The enzyme
was digested and cross-linked as described in the legend to Fig. 2. Samples were resolved on 16.5% Tricine gels. Only the
lower portions of the gels are shown. Lane 1, digested enzyme
prior to cross-linking. Lane 2, digested enzyme after
cross-linking. Lane 3, molecular mass markers (kDa): 27.5,
18.5.
These
findings clearly indicate the proximities of the 22-kDa C-terminal and
the 11-kDa N-terminal segments of the -subunit to each other, and
to the
-subunit.
When the enzyme that was
partially cleaved in this manner was solubilized and cross-linked, two
cross-linked products were detected (Fig. 4A, lane 4):
the expected ,
-dimer, and a band with mobility consistent
with that of a dimer of
and the 83-kDa C-terminal fragment of
(83,
-dimer). Note that on these 5% gels used to separate the
two cross-linked products,
and the 83-kDa monomer move together
as a diffuse band (Fig. 4A, lanes 3 and 4).
The two uncross-linked peptides, however, may easily be separated on
7.5% gels(28) .
For further identification of the presumed
83,-dimer, gels similar to those of Fig. 4A, lane
4, were immunostained with several antibodies. As expected, the
product reacted with the
-specific antibody (Fig. 4B) and the antibody against residues
1003-1013 of
(not shown). More importantly, the presumed
83,
-dimer did not react with antibodies against the 111-122
sequence of
(Fig. 4C) and the 16-30
sequence of
(not shown); suggesting that it did not include small
fragments containing the epitopes of these antibodies. When the
presumed 83,
-dimer was subjected to N-terminal analysis, only two
amino acid sequences were clearly identified (Table 1),
confirming its identity as the 83,
-dimer. There was no evidence
for the existence of other peptides in the cross-linked product. These
findings, in conjunction with the results of the experiments on the
enzyme extensively digested by trypsin ( Fig. 2and Fig. 3, and Table 1), indicate the direct contact of
with the C-terminal segment of
, and clarify the nature of
interactions involved in 11,22,
-trimer formation (see
``Discussion'').
Chemical cross-linking techniques that have been used
extensively in studies on the quaternary structure of
Na/K
-ATPase have suggested the
existence of
,
-, and
,
-interactions in the
membrane-bound enzyme(23) . The
,
-interactions are
not noted in cross-linking experiments with most detergent-solubilized
preparations of the enzyme(1, 2, 23) .
Because of this, and the well known difficulty of distinction between
stable and collision complexes within the membrane phase(42) ,
there have been disagreements on the meanings of the cross-linked
,
-complexes of the
enzyme(1, 2, 23) . There are no such
ambiguities, however, regarding
,
-associations. In
cross-linking experiments with preparations that are solubilized with a
variety of detergents, the
,
-dimer is the major or the only
product(1, 2, 23, 36, 37, 38) .
It is reasonable, therefore, to assume that this product represents the
stable noncovalent
,
-complex, and that localization of the
domains involved in oxidative cross-linking of
to
identifies some, if not all, of the
,
-interfaces of the
native enzyme.
The original intent of this work was to use
preparations with intact and defined proteolytic fragments of
to identify the regions of
that are in direct contact with
. In the process, it became apparent that the same approach also
provided information about the domains of
that are in contact
with each other within a single
chain. As in the case of the
cross-linked
,
-dimer obtained in detergent-solubilized
preparations, it is reasonable to assume that cross-linked products
containing
fragments also do not result from the random
collisions of the reactants.
The results of the experiments on the
preparation extensively digested by trypsin are more informative.
Formation of the cross-linked 11,22-dimer ( Fig. 3and Table 1) clearly indicates the proximities of the sulfhydryl
groups of these peptides. Although the C-terminal residue of the 11-kDa
peptide is not known, its N-terminal residue (Asp), and
its apparent molecular mass indicate that this peptide contains the
first two transmembrane helices of
(H
-H
),
and three cysteines (residues 86, 104, and 138) either within or close
to the transmembrane segments. The 22-kDa peptide is the intact
C-terminal segment of
containing four cysteines (residues 911,
930, 964, and 983). There are uncertainties regarding the structural
organization and the number of transmembrane segments in this part of
(3, 13, 14, 15, 30) .
However, if we assume the most likely 10-span model of
(3) , the four cysteines of the 22-kDa peptide would all
be within or adjacent to the three transmembrane segments
H
, H
, and H
. It is reasonable to
conclude, therefore, that the formation of cross-linked 11,22-dimer is
indicative of close contact between at least one N-terminal helix and
one C-terminal helix of
. Based on the estimated relative
positions of cysteine residues within these helices (3, 22) , two pairs of interacting helices involved in
cross-linking are suggested: H
-H
(Cys
and Cys
) and H
-H
(Cys
and Cys
). It is likely that the
disulfide cross-linking of the same helices is also involved in the
formation of 48,64-dimer (Fig. 1), and in the change of mobility
of the intact
monomer after cross-linking (Fig. 1). To our
knowledge, these are the first experimental data to provide concrete
information on three-dimensional helix-helix interactions of the
enzyme. Efremov et al.(20, 21, 22) have addressed the
important issue of the spatial organizations of these helices using
theoretical and molecular modeling methods. Studies toward the more
precise identifications of the cross-linked residues and helices are in
progress.
Which sulfhydryl(s)
cross-links to a sulfhydryl(s) of the 22-kDa segment of
? The
-subunit contains a cysteine (Cys
) within its only
transmembrane segment, and three cystines within its extracellular
domains(3, 44) . If the cysteines of the 22-kDa
fragment of
are within the H
-H
intramembrane domains (as indicated by the favored 10-span model
of
), close contact must be between one of these segments and the
one intramembrane segment of
. Because of uncertainties on the
topology of the C-terminal region of
, however, cross-linkings of
extracellular domains of
to those of the 22-kDa segment of
cannot be ruled out.
An important issue is the relation of the
present findings to the conclusions of previous studies on the
structural requirements for the assembly of
Na/K
-ATPase. Several elegant studies
on the expressions of
,
, and their mutants in mammalian
cells and Xenopus oocytes have provided considerable
information on the structural elements of the two subunits that are
required for the assembly of the
,
-complex(11, 15, 16, 17, 18, 19) ;
and have shown the importance of both the transmembrane and the
extracellular domains of
for efficient assembly(19) . It
is appropriate to note two points about these studies: in principle,
subunit interactions that are essential to the processes involved in
the assembly need not be the same as those of the assembled enzyme of
the plasma membrane. Also, while studies on assembly identify domains
and residues that are important to
,
-contacts necessary for
assembly, they do not distinguish those structural elements that
regulate the interface from those that are at the interface. On the
other hand, while the present cross-linking experiments reveal some
domains and residues that are at interfaces, they do not identify all
relevant interfaces. Clearly, these and other experimental approaches
have the potential of complementing each other.