(Received for publication, July 9, 1996, and in revised form, November 7, 1996)
From the An immunofluorescence study of adult rat muscle
tissues with a polyclonal antibody against the RGD-directed fibronectin
receptor of Friend's erythroleukemia cells
( Skeletal muscle is a specialized tissue converting chemical energy
into mechanical work. For this reason, both the endo- and exosarcomeric
compartments are equipped with a very well developed cytoskeleton,
ensuring structural stability to the sarcomeres and to the myofibrils
(1). In addition, skeletal muscle is equipped with a complex meshwork
of skeletal muscle proteins, ensuring mechanical stability to the
sarcolemma. Mechanical stress of membrane cytoskeletal actin filaments
can be discharged to the extracellular matrix in at least two ways: the
first is represented by the In recent years, it has become clear that integrins play a major
role in mediating interactions of muscle cells with the extracellular matrix and in muscle differentiation. Integrin receptors are
heterodimeric transmembrane glycoproteins composed of Studies of the Several integrins of the To address the role of This study was carried out on male rats (albino Wistar) and
rabbits (New Zealand) fed ad libitum with standard
laboratory food and tap water.
A rabbit polyclonal antibody against the
RGD-directed fibronectin receptor of Friend's murine erythroleukemia
cells ( Antibodies reactive with the 167-kDa protein were purified from the
anti- A polyclonal antiserum against the peptide
X-Leu-Gln-Val-Thr-Trp-Arg-Ile-Thr-Asn (Chiron Mimotopes Pty.
Ltd.) (a 167-kDa protein internal sequence) was raised in New Zeland
White rabbits by subcutaneous injections. For the first injection, we
used 330 µg of peptide solubilized in PBS (0.15 M NaCl,
0.1 M Na2HPO4, and 0.1 M NaH2PO4, pH 7.2) and mixed 1:1
(v/v) with Freund's complete adjuvant. Rabbits were boosted twice at
~4-week intervals and six times at ~2-week intervals with 330 µg
of the peptide mixed 2:1 (v/v) with Freund's incomplete adjuvant.
Samples of soleus muscles were
prepared from 3-4-month-old Wistar rats. Frozen sections (8-10 µm
thick) were allowed to attach to glass slides treated with 2% gelatin
in PBS. After saturation with preimmune serum for 30 min, the sections
were incubated for 30 min with the antibody (50 µg/ml in PBS), washed
thoroughly in PBS, and reacted with fluorescein isothiocyanate-labeled
goat anti-rabbit IgG (Sigma) diluted 1:10,000 in PBS. All incubations were carried out at room temperature.
A membrane fraction from rat or
rabbit fast-twitch muscle, kidney, and liver was prepared as follows.
The minced tissues were passed in a Potter-Elvehjem homogenizer with a
tight-fitting, rotating Teflon pestle in 0.3 M sucrose, 5 mM imidazole, and 5 mM sodium EGTA, pH 7.0 (~4 ml of solution were used per g of tissue). The homogenates were
centrifuged at 6500 × gmax for 20 min in a
refrigerated Sorvall RC5B centrifuge. The pellets were discarded, and
the supernatants were centrifuged at 15,000 × gmax for 30 min. The supernatants were spun at
150,000 × gmax for 60 min in a Beckman L70
ultracentrifuge. The resulting membrane pellets were resuspended in
isolation buffer at ~5 mg/ml, frozen in liquid nitrogen, and stored
at Terminal cisternae from rabbit fast-twitch muscle sarcoplasmic
reticulum were prepared by the following method. Briefly, 200-220 g of
rabbit back and hind leg muscles were homogenized in 900 ml of 0.3 M sucrose and 5 mM imidazole, pH 7.4, in a
Waring blender for 2 min. The homogenate was centrifuged at 7700 × gmax for 20 min. The supernatant was
discarded, and the homogenization and centrifugation steps were
repeated for the pellet. The resulting supernatant was filtered through
three layers of cheesecloth and centrifuged at 110,000 × g for 90 min in the Beckman ultracentrifuge. The pellet was
resuspended in 30 ml of 2 M sucrose and 5 mM
imidazole, pH 7.4; placed at the bottom of Ti-60 centrifuge tubes; and
overlaid with a discontinuous sucrose gradient consisting of 1.32, 1.11, 0.90, and 0.79 M sucrose in 5 mM
imidazole, pH 7.4 (5 ml/step). After centrifugation at 150,000 × g for 90 min, the membranes banding at each interface were
collected; diluted in 5 mM imidazole, pH 7.4; and
centrifuged at 150,000 × g for 45 min. Each pellet was
resuspended in 0.3 M sucrose and 5 mM
imidazole, pH 7.4; frozen in liquid nitrogen; and stored at For some experiments, a fraction enriched in the 167-kDa protein was
prepared as follows. Rabbit slow-twitch (soleus) muscles (~10 g) were
homogenized using a Teflon-glass homogenizer in 50 ml of 20 mM NaH2PO4, 20 mM
Na4P2O7, 0.3 M KCl, 1 mM MgCl2, 0.5 mM EDTA, and 1 mM EGTA, pH 7.0, for 20 s. The homogenate was
centrifuged at 16,300 × g for 20 min. The supernatant
was discarded, and the pellet was homogenized in 40 ml of 5 mM histidine and 0.1 mM EGTA. The homogenate
was centrifuged at 40,000 × g for 30 min. The
supernatant was discarded, and the pellet was resuspended in 0.6 M KCl, 5 mM histidine, 0.3 M
sucrose, and 0.1 M EGTA and then centrifuged at 40,000 × g for 30 min. The final supernatant was frozen in liquid
nitrogen and stored at Terminal cisternae
membrane fractions from rabbit fast-twitch skeletal muscle were
prepared as described above. Approximately 200 mg of terminal cisterna
membranes in 20 ml of 0.3 M sucrose and 5 mM
imidazole, pH 7.4, were diluted in 250 ml of 0.6 M NaCl, 20 mM Na2HPO4, and 20 mM
NaH2PO4, pH 7.4, and gently stirred at 4 °C
for 30 min. The suspension was then centrifuged at 150,000 × gmax for 30 min. The resulting pellet, which was
largely depleted of myosin, was resuspended in 20 ml of 20 mM Na2HPO4 and 20 mM NaH2PO4, pH 7.4. After bringing the pH to 11.0 with 1 N NaOH, the suspension was gently stirred for 30 min
at 4 °C and ultracentrifuged as described above. The supernatant,
which contained all of the 167-kDa protein, was brought to pH 7.4 with
1 N HCl, made 0.5 M in NaCl and 0.5% (w/v) in
CHAPS, and further fractionated by chromatography on
IDA-Zn2+-agarose (Sigma) as described below.
Ten milliliters of IDA-Zn2+-agarose matrix were
equilibrated with 10 volumes of acetate buffer (0.1 M
sodium acetate, pH 4.5) followed by 5 volumes of 10 mM
ZnCl2 in acetate buffer and by at least 10 volumes of
acetate buffer to remove the excess zinc. After equilibration of the
column with at least 20 volumes of binding buffer (0.5 M
NaCl, 20 mM Na2HPO4, 20 mM NaH2PO4, and 0.5% (w/v) CHAPS,
pH 7.4), the neutralized supernatant from the last centrifugation step
(see above) was loaded onto the column, and the flow-through fraction
was reapplied for five times. The flow-through fraction, which
contained most of the 167-kDa protein, was dialyzed against 100 mM NaCl, 10 mM Tris, pH 8.5, 2 mM
dithiothreitol, and 0.1 mM PMSF. A visible precipitate
formed, which was collected by centrifugation and which contained the
167-kDa protein virtually free of sarcalumenin (see Fig. 4).
SDS-PAGE (5-10% acrylamide
continuous gradient gel, 50 µg of protein/lane) was carried out as
described (23). The gels were stained with either Coomassie Brilliant
Blue or Stains-all (Sigma) as described (24). For Western blotting,
proteins were transferred to nitrocellulose sheets (Schleicher & Schuell, Dassel, Federal Republic of Germany) for 4 h at 1 A with
a Hoefer apparatus at 4 °C (28). The nitrocellulose was stained with
Ponceau Red (0.2%, w/v) in 3% (v/v) trichloroacetic acid,
photographed, destained in H2O, and finally blocked for
1 h at room temperature in Tris-buffered saline containing 10%
(v/v) defatted milk (blocking buffer). The blocking solution was
discarded, and the nitrocellulose was incubated with (i) 10 µg/ml
purified immune IgG, (ii) a 1:5 dilution of the polyclonal antiserum
against the 167-kDa peptide, (iii) the anti- Electrofocusing was
carried out on 6% polyacrylamide capillary gels containing 2%
Ampholine (Pharmacia Biotech AB; pH interval of 3.5-10). The second
dimension was SDS-PAGE on 5-10% acrylamide continuous gradient slab
gels.
The proteins were electroblotted onto
polyvinylidene difluoride membrane in a semidry apparatus (Hoefer)
using a Towbin (28) buffer (50 mM Tris-HCl, 192 mM glycine, 0.02% (w/v) SDS, 10% (v/v) methanol, and 2 mM dithiothreitol) for 1 h at ~1.2
mA/cm2. The transferred protein was visualized on the
membrane by a 5-min incubation in 0.1% (w/v) Serva blue R in 50%
(v/v) methanol, followed by destaining in 70% (v/v) methanol for 5-10
min. The dried polyvinylidene difluoride membrane slices were cut into small pieces (~1 mm2) and equilibrated for 1 h at
room temperature in 10 µl of 1% (w/v) Normal rat kidney fibroblasts
were grown to 90% confluency in Dulbecco's modified Eagle's medium
(Poiesys, Padova, Italy) supplemented with 10% heat-inactivated fetal
calf serum, 20 mM glutamine, and penicillin/streptomycin in
a CO2 incubator. Cells in 75-cm2 tissue culture
flasks (Greiner) were washed twice with ice-cold PBS and then extracted
on ice with 1 mM CaCl2, 1 mM
MgCl2, 10 mM Tris/MOPS, pH 7.4, 1 mM PMSF, and 0.2 M To study the expression of
To identify the protein(s) responsible for the fluorescence labeling, a
Western blot analysis of total membrane fractions from rat soleus
muscle was carried out. Fig. 1C shows that the 167-kDa
protein was recognized in rat soleus muscle (lane 1), but
not in kidney (lane 2). As described above, the overall
pattern of antigen expression in rat muscle suggested an intracellular distribution of the 167-kDa protein. This finding is strengthened by
the fact that a protein of the same size was recognized by this rabbit
antibody in rabbit muscles. Indeed, a 167-kDa protein was revealed in
heart (lane 3), gastrocnemius (lane 4), soleus (lane 5), and psoas (lane 6) muscles from rabbit.
The reaction with this protein was specific since an irrelevant rabbit
antiserum gave no detectable signal (lanes 7-10), while the
intensely stained band at ~70 kDa was also detected by an irrelevant
rabbit serum and is therefore not specific.
Fig. 1D illustrates the pattern of distribution of the
167-kDa protein in rabbit tissues. The protein was detected in a total homogenate from skeletal muscle (lane 1), but not from liver
(lane 2) or kidney (lane 3). The subcellular
distribution of the 167-kDa protein in skeletal muscle was investigated
further. It can be seen that the protein was not present in a cytosolic
fraction (lane 4), while it was clearly detected in a total
membrane fraction (lane 5), in purified sarcolemma
(lane 6), and in the terminal cisternae of the SR
(lane 7). This experiment clearly indicates that
the 167-kDa protein is membrane-associated.
To investigate the nature of the interactions of the 167-kDa protein
with muscle membranes, a fraction enriched in terminal cisternae was
exposed to increasing pH. The solubilized proteins and the membranes
were then separated by either ultracentrifugation or filtration through
0.45-µm pore-sized filters, and each fraction was separated by
SDS-PAGE and probed with the anti-receptor antibody after transfer to
nitrocellulose filters. Fig. 2 shows the Western blot of
such an experiment. The 167-kDa protein was membrane-associated at pH
lower than 9, was partially solubilized at pH 9, and was completely
recovered in the soluble fraction at pH 10 or higher. These findings
indicate that the 167-kDa protein is a peripheral membrane protein. To
obtain information about its location, we subjected a terminal
cisternae membrane fraction to controlled proteolysis with trypsin in
the absence or presence of Triton X-100. After blocking the reaction by
the addition of soybean trypsin inhibitor and boiling in SDS gel sample
buffer (21), Western blotting with the anti-receptor antibody was
carried out after SDS-PAGE separation and transfer. Fig. 2B
shows that the 167-kDa protein (arrow) could be digested by
trypsin (lanes 3 and 4) only in the presence of
detergent (lane 4) and that the detergent per se
had no effect on the reactivity of the protein (lanes 1 and
2).
In the molecular mass region of 165-170 kDa, SR terminal cisternae are
enriched in two proteins, the 165-kDa sarcalumenin (22, 23) and the
170-kDa histidine-rich protein (24, 25). Fig.
3B shows an SDS-PAGE separation of proteins
from SR terminal cisternae, stained with Stains-all. Three polypeptides
can be clearly identified in the 165-170-kDa range. Only the lower and higher molecular mass species underwent a metachromatic shift, while
the intermediate-sized protein (arrow) maintained the
original pink stain. Fig. 3B shows that the 167-kDa protein
can be easily distinguished from histidine-rich protein and
sarcalumenin on two-dimensional gels, owing to its slightly higher
pI.
We have further purified the 167-kDa protein from SR terminal cisternae
by extraction at pH 11 (see "Experimental Procedures" for details).
The extract, containing histidine-rich protein, sarcalumenin, and the
167-kDa protein (data not shown), was neutralized and further
fractionated through an IDA-Zn2+-agarose column. As
expected (26), sarcalumenin was recovered in the unbound fraction
together with most of the 167-kDa protein, while histidine-rich protein
remained bound to the column and could only be eluted with 0.1 M EDTA (data not shown). The flow-through fraction of the
IDA-Zn2+-agarose column was finally dialyzed against 100 mM NaCl, 10 mM Tris, pH 8.5, 2 mM
dithiothreitol, and 0.1 mM PMSF; and under these
conditions, a precipitate formed that contained the 167-kDa protein,
but not sarcalumenin, as judged by SDS-PAGE (Fig. 3D) and
two-dimensional electrophoresis (Fig. 3C). Note that only one protein of 167 kDa could be identified, which did not undergo a
color shift with the Stains-all dye (arrows). Western
blotting of this preparation confirmed its identity to the 167-kDa
protein (data not shown).
We next used two-dimensional gel separation followed by transfer to
nylon membranes for sequencing the N terminus of the 167-kDa protein,
which yielded the sequence
X-X-Phe-Gly-Tyr-Thr-Gly-Leu, and a trypsin
cleavage fragment, which yielded the sequence
X-Leu-Gln-Val-Thr-Trp-Arg-Ile-Thr-Asn. Surprisingly, these
sequences did not match any known integrins or any sequence in the
Swiss and EMBL data bases. These findings indicate that 167-kDa protein
is a novel, muscle-specific protein of the SR terminal cisternae that
has probably escaped attention so far because of its comigration with
sarcalumenin.
To understand the basis for its apparent cross-reactivity with the
anti- Fig. 4A shows that while the
anti- The basis for the reactivity of our anti-integrin antiserum with the
167-kDa protein remains obscure. One possibility is that a protein
related to the 167-kDa protein or an ~140-kDa proteolytic product was
present in the material used for immunization. Contamination by the
"native" 167-kDa protein appears extremely unlikely because (i) the
integrin was purified by sequential chromatographic steps on wheat germ
agglutinin and DEAE-cellulose (neither of which binds the 167-kDa
protein) prior to affinity chromatography on a fibronectin peptide and
elution with GRGDSP (18), and (ii) only a single diffuse band of ~140
kDa could be detected by silver staining after SDS-PAGE separation of
the material used for immunization under reducing conditions (data not
shown, but see Ref. 18). However, integrin recognition motifs have been
found in several intracellular muscle proteins, including the
N-terminal REDV sequence of dystrophin (17), and in SR intraluminal
proteins, as in the case of
The function of the 167-kDa protein remains to be elucidated and can
only be a matter of speculation at present. Its muscle-specific expression and subcellular distribution, however, suggest a role in
muscle cell physiology, which could be related to regulation of
Ca2+ homeostasis. Determination of the primary sequence of
the 167-kDa protein is under way in our laboratories and should
contribute to resolving the quest for its function.
Department of Biomedical Sciences,
Biomembranes,
5
1-integrin) unexpectedly revealed a
pattern of intracellular antigen distribution. Western blotting
analysis of rat and rabbit membrane fractions indicated that the
antibody recognizes a 167-kDa protein expressed both in heart and in
skeletal muscle (relative abundance: heart > slow muscle > fast muscle), but not in liver and kidney. The 167-kDa protein did not
show altered electrophoretic mobility upon reduction and failed to bind
several lectins, including wheat germ agglutinin. A study of its
subcellular distribution in rabbit skeletal muscle revealed that the
167-kDa protein is mostly associated with the terminal cisternae of the
sarcoplasmic reticulum (SR) and, to a smaller extent, with the
sarcolemma, while it is absent in the longitudinal tubules of the SR.
The 167-kDa protein is not an integral membrane protein since it can be
extracted at pH
10. This protein can be proteolytically cleaved only
in the presence of detergent, indicating that it resides on the luminal
side of the SR. The 167-kDa protein could be resolved from the closely spaced sarcalumenin and histidine-rich protein by column chromatography followed by detergent dialysis and two-dimensional gel electrophoresis. The N terminus and the internal sequences did not match any known sequence in protein and DNA data bases, indicating that the 167-kDa protein is a novel muscle protein selectively localized to the SR.
Integrins from rat kidney fibroblasts were not recognized by either (i)
a polyclonal antiserum against the purified 167-kDa protein or (ii) the
anti-
5
1-integrin antiserum after affinity purification onto the 167-kDa protein. These data indicate that the
167-kDa protein is not immunologically cross-reactive with integrins,
despite its reaction with a polyclonal anti-integrin antibody.
- and
-dystroglycan complex, which
binds dystrophin and actin-binding protein at the cytoplasmic side of
the sarcolemma and merosin at the extracellular matrix side; the second
is represented by vinculin (2), talin, spectrin, and ankyrin localized
to the costameres (3, 4).
- and
-subunits. Several isoforms of both
- and
-subunits have been
characterized that are apparently expressed at different times in the
process of muscle development (5).
1-subunit revealed that it is localized
to the myotendinous and neuromuscular junctions and to the costameres (6, 7). The importance of the structural role of
1-integrin was demonstrated in studies of
Drosophila mutants lacking
1-subunit expression: the mutant showed an abnormal muscle tissue morphology, without proper organization of myofibers and the myotendineus junction
(8).
1-subfamily have now been shown
to play a role in muscle physiology during development, including
4
1 and its counter-receptor vascular cell
adhesion molecule-1 (9, 10),
5
1 (11, 12),
7
1 (13, 14), and
9
1 (15). In particular, the
5
1-integrin is involved in the adhesion
of muscle to the extracellular matrix and has been shown to colocalize with dystrophin in the adhesion plaque-like structures of myoblasts and
myotubes (16). This integrin connects the extracellular matrix to the
cytoskeleton because it binds fibronectin outside the cell and talin
and
-actinin inside the cell. It is also intriguing that integrin
recognition motifs are also present in several intracellular muscle
proteins, including the N terminus REDV sequence of dystrophin (17).
1-integrins in muscle physiology,
we carried out a screening of adult rat muscle tissues with a
polyclonal antibody against the RGD-directed fibronectin receptor of
Friend's erythroleukemia cells
(
5
1-integrin). Unexpectedly, we detected a pattern of intracellular antigen distribution. Western blotting analysis indicated that the antibody recognized a 167-kDa protein expressed both in heart and in skeletal muscle, but not in liver and
kidney. The 167-kDa protein is a peripheral membrane protein mostly
associated with the luminal side of the terminal cisternae of the
sarcoplasmic reticulum (SR)1 and, to a
smaller extent, with the sarcolemma, while it is absent in the
longitudinal tubules of the SR. The 167-kDa protein could be resolved
from the closely spaced sarcalumenin and histidine-rich protein by
column chromatography followed by detergent dialysis and
two-dimensional gel electrophoresis. Partial protein sequences revealed
a unique sequence that is not present in protein or DNA data bases,
indicating that the 167-kDa protein is a novel muscle protein
selectively localized to the SR. Indeed, rat fibroblast integrins were
not recognized by either (i) a polyclonal antiserum against the peptide
X-Leu-Gln-Val-Thr-Trp-Arg-Ile-Thr-Asn (the inner sequence of
the 167-kDa protein) or (ii) the
anti-
5
1-integrin antiserum after affinity
purification onto the 167-kDa protein.
5
1-integrin) was prepared as
described (18). An IgG fraction of the immune serum was prepared by
protein G-Sepharose affinity chromatography (Pharmacia Biotech Inc.)
and used in most of the experiments described in this study, as
specified in the figure legends. This antibody has features similar to
those of a previously described antibody (data not shown, but see Ref.
18).
5
1-integrin antiserum by absorption
onto immobilized 167-kDa protein obtained from two-dimensional gel
separation and transfer onto nitrocellulose. The nitrocellulose strips
containing the 167-kDa protein were incubated overnight at 4 °C with
the anti-
5
1-integrin antiserum diluted
1:100 in Tris-buffered saline (0.15 M NaCl and 20 mM Tris-HCl, pH 7.5). The nitrocellulose was washed three
times with 0.15 M NaCl, once with 0.5 M NaCl
and 0.1% Tween 20, and again three times with 0.15 M NaCl
(5 min/wash). Antibodies reactive with the 167-kDa protein were
detached by treatment with 0.2 M glycine, pH 2.8, for
30 s, and neutralized with 2 M Tris-HCl, pH 8, to a
final pH of 7.4 after transfer to a fresh test tube.
80 °C.
80 °C.
The 0.79-0.90 M sucrose interface is enriched in
T-tubules; the 0.90-1.11 M sucrose interface is enriched
in longitudinal reticulum; the 1.11-1.32 M sucrose interface contains a mixture of longitudinal reticulum and terminal cisternae; and the 1.32-2 M sucrose interface is highly
enriched in terminal cisternae of the sarcoplasmic reticulum. Triads
were purified exactly as described (19); resuspended in 0.3 M sucrose and 5 mM Hepes, pH 7.1; frozen in
liquid nitrogen; and stored at
80 °C. The isolation of the
sarcolemma was carried out as described by Luise et al.
(20). All buffers contained 0.23 mM PMSF, 0.83 mM benzamidine, 1 mM iodoacetamide, 1 mM leupeptin, 0.7 mM pepstatin, and 76.8 nM aprotinin.
80 °C.
Fig. 4.
Reactivity of fibroblast integrins and the
167-kDa protein with 5
1-integrin and
167-kDa peptide antisera. A, extracts from normal rat kidney
fibroblasts (lanes 1 and 3) and membrane fractions enriched in terminal cisternae from rabbit fast-twitch skeletal muscle SR (lanes 2 and 4) were separated
by SDS-PAGE, transferred to nitrocellulose, and blotted with (i)
anti-
5
1-integrin antibody at a 1:100
dilution (lanes 1 and 2) or (ii) antibodies purified from the same serum onto immobilized 167-kDa protein (lanes 3 and 4). B, extracts from
normal rat kidney fibroblasts (lanes 1 and 3) and
fractions enriched in the 167-kDa protein from rabbit slow-twitch
skeletal muscle (lanes 2 and 4) were separated by
SDS-PAGE, transferred to nitrocellulose, and blotted with (i) a
polyclonal antiserum against the 167-kDa peptide (lanes 1 and 2) or (ii) an irrelevant antiserum (lanes 3 and 4) at a 1:100 dilution. The positions of
1-integrin (
) and the 167-kDa protein (
) are
indicated. Note that the 167-kDa protein-specific antibodies do not
cross-react with
1-integrin, but only recognize the
167-kDa protein.
[View Larger Version of this Image (11K GIF file)]
5
1-integrin antiserum after affinity
purification onto the 167-kDa protein (see above), or (iv) a 1:100
dilution of anti-
5
1-integrin or
irrelevant rabbit antiserum (see figure legends for details). The
secondary antibody used was a goat anti-rabbit IgG conjugated with
alkaline phosphatase (Sigma) or with peroxidase (Calbiochem) at the
recommended dilution (1:20,000 and 1:5000, respectively) in blocking
buffer. After removing the antibody solution, the blots were rinsed
four times with Tris-buffered saline and 0.05% Tween 20 and finally
developed with nitro blue tetrazolium or with Chemiluminescence
(Boehringer Mannheim).
-octyl glucopyranoside and
100 mM ammonium bicarbonate, pH 7.8. Digestion was
initiated by the addition of 1 µg of trypsin (Promega) in 1 µl of
the same buffer and was carried out for 15 h at room temperature.
Digestion was stopped by the addition 1 µl of 2% (v/v)
trifluoroacetic acid to the sample. The supernatant was collected;
membrane pieces were washed twice with 10 µl of 0.1% trifluoroacetic
acid; and each wash was pooled with the supernatant. The peptide
mixture was resolved by reverse-phase high pressure liquid
chromatography on a capillary column (C18, 5 µm, 300 Å, 280 × 0.32 mm; LC Packings International, Zürich). The
total flow was 3 µl/min, and the column was washed extensively with solvent system A (0.1% (v/v) trifluoroacetic acid in H2O)
before running a 60-min linear gradient from 0 to 70% solvent system B
(80% (v/v) acetonitrile and 0.08% (v/v) trifluoroacetic acid). Peptides were collected directly onto trifluoroacetic acid-treated glass fiber discs that had been precoated with Polybrene. These were
analyzed on an Applied Biosystems 476A Protein Sequencer, with
sequencing cycles being run according to standard protocols provided by
the manufacturer.
-octyl glucopyranoside (2 ml of extraction buffer/flask). After 20 min, the bottoms of the flasks
were carefully scraped with a rubber policeman, and the extract was
transferred to polypropylene test tubes. After a further 20 min on ice,
the extracts were cleared by centrifugation at 15,000 × gmax in an Eppendorf microcentrifuge. Samples
were boiled with an equal volume of double-strength Laemmli gel sample buffer (21) under nonreducing conditions and subjected to SDS-PAGE and
Western blotting as described above.
1-integrins in adult rat
tissues, we screened a series of frozen sections with a rabbit
polyclonal antibody against the fibronectin receptor
(
5
1-integrin) of Friend's erythroleukemia cells (18). No specific staining beyond the vascular
compartment was seen in liver and kidney (data not shown), while a
specific reaction was revealed with soleus muscle (Fig. 1) and with heart and extensor digitorum longus muscles
(data not shown). It can be seen that besides the intense reactivity of
endothelial cells in the capillaries surrounding the muscle fibers, the
fluorescence displayed a clear reactivity with the muscle fibers
themselves (Fig. 1B). A prominent banding pattern with
periodicity of ~1.3-1.5 µm could be observed, while no reactivity was detected with the secondary antibody alone (Fig. 1A).
These experiments indicate that the antibody recognizes a
muscle-specific antigen that appears to be expressed both in heart and
in slow and fast skeletal muscle. The overall pattern of fluorescence distribution suggests, unexpectedly, that the antigen may be localized within either the T-tubules or the terminal cisternae.
Fig. 1.
Reactivity of rat and rabbit tissues with a
mouse anti-fibronectin receptor
(5
1-integrin) antibody. A and
B, 10-µm-thick frozen sections from soleus muscle were
reacted with the anti-receptor antibody followed by a secondary goat
anti-rabbit IgG fluoresceinated antibody (B) or by the
secondary antibody alone (A) as described under
"Experimental Procedures" and visualized by fluorescence microscopy. Bar = 32 µm. C, Western blot
analysis was carried out on total membrane fractions from rat soleus
muscle (lane 1) and kidney (lane 2) and from
rabbit heart (lanes 3 and 7), gastrocnemius (lanes 4 and 8), psoas (lanes 5 and
9), and soleus (lanes 6 and 10)
muscles with the anti-receptor antibody (lanes 1-6) or with an irrelevant rabbit antiserum (lanes 7-10). Molecular mass
standards (bars) were rabbit muscle myosin (205 kDa),
Escherichia coli
-galactosidase (116 kDa), rabbit muscle
phosphorylase b (97.4 kDa), and bovine serum albumin (66 kDa). D, Western blot analysis was carried out on rabbit
tissues with the anti-receptor antibody. Lane 1, skeletal muscle homogenate; lane 2, liver homogenate; lane
3, kidney homogenate; lanes 4-7, skeletal muscle
subcellular fractions: cytosol, total membranes, sarcolemma, and
terminal cisternae, respectively.
[View Larger Version of this Image (64K GIF file)]
Fig. 2.
Extraction of the 167-kDa protein at alkaline
pH and trypsin sensitivity in the presence of detergent. A,
membrane fractions enriched in terminal cisternae from rabbit
fast-twitch skeletal muscle were incubated in 0.3 M
sucrose, 5 mM imidazole, 5 mM EDTA, 5 mM EGTA, and NaOH to give a final pH of 8, 9, 10, or 11. After incubation for 30 min at 0 °C with constant stirring, a
soluble and a membrane fraction were prepared by centrifugation at
200,000 × gmax for 60 min. Each fraction
was separated by SDS-PAGE, transferred to nitrocellulose, and probed
with the anti-receptor antibody. s, supernatant;
p, pellet. Lanes 1 and 2, pH 8;
lanes 3 and 4, pH 9; lanes 5 and
6, pH 10; lanes 7 and 8, pH 11;
lane 9, untreated SR terminal cisternae fraction.
B, duplicate aliquots of the same SR terminal cisternae
fraction were incubated with (lanes 3 and 4) or
without (lanes 1 and 2) trypsin (80:1 ratio to
trypsin in weight) in 0.3 M sucrose and 5 mM
imidazole, pH 7.4, in the presence (+; lanes 2 and
4) or absence (; lanes 1 and 3) of
1% (v/v) Triton X-100. After 3 min at room temperature, all reactions
were transferred on ice, and soybean trypsin inhibitor (2:1 ratio to
trypsin) was added, immediately followed by solubilization in SDS and
separation by SDS-PAGE. After transfer to nitrocellulose, the proteins
were stained with Ponceau Red, destained, and probed with the
anti-receptor antibody.
[View Larger Version of this Image (44K GIF file)]
Fig. 3.
SDS-PAGE and two-dimensional separation of
terminal cisterna proteins. A and B, an SR
terminal cisternae fraction was separated by SDS-PAGE (B) or
by isoelectrofocusing followed by SDS-PAGE (A) and stained
with Stains-all dye. Note that a non-metachromatic 167-kDa protein
() with a slightly higher isoelectric point can be resolved from the
closely spaced histidine-rich protein (
]) and sarcalumenin (*).
C and D, a pH 11 extract from a rabbit SR terminal cisternae fraction was neutralized and fractionated by chromatography on an IDA-Zn2+-agarose column. The
flow-through fraction, containing most of the 167-kDa protein and
sarcalumenin, was dialyzed against 100 mM NaCl, 10 mM Tris, pH 8.5, 2 mM dithiothreitol, and 0.1 mM PMSF. A visible precipitate formed, which was collected
by centrifugation and separated by SDS-PAGE (D) or by
isoelectrofocusing followed by SDS-PAGE (C) and stained with
Stains-all dye. Note the substantial enrichment of the
non-metachromatic 167-kDa protein (
) and the absence of sarcalumenin
(compare with B).
[View Larger Version of this Image (86K GIF file)]
5
1-integrin antiserum, we followed
two strategies. In the first, we prepared rabbit antibodies against the
synthetic peptide X-Leu-Gln-Val-Thr-Trp-Arg-Ile-Thr-Asn,
modeled on the 167-kDa protein unique sequence. In the second, we used
immobilized 167-kDa protein as an affinity matrix to purify specific
antibodies from the anti-
5
1-integrin
antiserum. We then used the anti-peptide and affinity-purified
antibodies to probe extracts from both rat 3T3 fibroblasts and a
167-kDa protein-containing muscle fraction in Western blots.
5
1-integrin antiserum recognized
both the
-integrin subunit of normal rat kidney fibroblasts (lane 1) and the 167-kDa protein in muscle (lane
2), the affinity-purified antibody failed to recognize
-integrin (lane 3), while, as expected, it still reacted
with the 167-kDa protein (lane 4). Likewise, the
anti-peptide antiserum recognized the 167-kDa protein in a membrane
fraction from rabbit slow-twitch skeletal muscle (Fig. 4B,
lane 2), but not
-integrin (lane 1). Note that
a preimmune serum did not stain either
-integrin (lane 3)
or the 167-kDa protein (lane 4). We must conclude that the
167-kDa protein and
-integrins are not antigenically related.
2
1-integrin-calreticulin interactions
(27).
*
This work was supported in part by the Consiglio Nazionale
delle Ricerche and the Ministero per l'Universitá e la Ricerca Scientifica e Tecnologica and by Telethon-Italy Grant 179 (to P. B.).
**
To whom correspondence should be addressed: Dipt. di Scienze
Biomediche Sperimentali, Via Trieste 75, I-35121 Padova, Italy. Tel.:
39-49-827-6025; Fax: 39-49-827-6049.
1
The abbreviations used are: SR, sarcoplasmic
reticulum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl
fluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1
propanesulfonate; IDA, iminodiacetic acid; PAGE, polyacrylamide gel
electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic
acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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