From the
The chicken gizzard smooth muscle extracellular ATPase
(ecto-ATPase) is a low abundance, high specific activity, divalent
cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The
ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout,
J.G. and Kirley, T.L.(1994) J. Biochem. Biophys. Methods 29,
61-75). In this study we evaluated the characteristics of a bank
of monoclonal antibodies raised against a partially purified chicken
gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase
capture assay were tested for effects on ATPase activity as well as for
their Western blot and immunoprecipitation potential. The five most
promising monoclonal antibodies were used to immunopurify the
ecto-ATPase. The one-step immunoaffinity purification of solubilized
chicken gizzard membranes with all five of these monoclonal antibodies
isolated a 66-kDa protein whose identity was confirmed by N-terminal
sequence analysis to be the ecto-ATPase. Several of these monoclonal
antibodies stimulated ecto-ATPase activity similar to that observed
previously with lectins. Western blot analysis revealed that three of
the five monoclonal antibodies recognized a major immunoreactive band
at 66 kDa (53-kDa core protein), consistent with previous purification
results. The other two antibodies recognized proteins of approximately
90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and
presumably associated or related) protein was identified by N-terminal
analysis as LEP100, a glycoprotein that shuttles between the plasma and
lysosomal membranes. The approximately 160-kDa co-immunopurifying
protein was identified by N-terminal analysis as integrin, a protein
involved in extracellular contacts with adhesion molecules. Extended
N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase
revealed some sequence homology with mouse lysosomal associated
membrane protein. Tissue distribution of the ecto-ATPase showed that
the highest levels of protein were expressed in muscle tissues
(cardiac, skeletal, and smooth) and brain.
Extracellular ATPases (ecto-ATPases)
The
purification and subsequent characterization of ecto-ATPases has been a
primary goal of our laboratory for several years. The first ecto-ATPase
purified to homogeneity was isolated from rabbit skeletal muscle
transverse tubule (t-tubule) membranes
(18) . The rabbit t-tubule
ecto-ATPase consists of a 67-kDa glycoprotein with a core protein of 52
kDa and a hydrophobic, proline-rich N terminus that is homologous to
the SH3 binding consensus sequence
(7) . The rabbit t-tubule
ecto-ATPase has a very high specific ATPase activity, but the
difficult, low yield, purification
(18) made obtaining
quantities of the t-tubule ecto-ATPase needed for structural studies
impossible. In a recent study, the chicken gizzard smooth muscle was
identified as an enriched source of ecto-ATPase
(2) . The chicken
gizzard smooth muscle ecto-ATPase was purified to homogeneity and
partially characterized
(8) . The enzyme consists of a 66-kDa
glycoprotein with a core protein of 53 kDa and a unique, hydrophobic N
terminus
(8) . Thus, the chicken smooth muscle (gizzard)
ecto-ATPase is very similar to the rabbit skeletal muscle t-tubule
ecto-ATPase. However, the catalytic activity of the chicken enzyme,
unlike the rabbit enzyme, is stimulated by lectins, including
concanavalin A
(8) . Although the purification strategy developed
for the chicken gizzard ecto-ATPase was successful
(8) , the
quantity of pure ecto-ATPase isolated was still not sufficient for
structural investigations.
This report describes our recent efforts
to generate and utilize monoclonal antibodies to characterize the
chicken ecto-ATPase. Previously, monoclonal antibodies were generated
against a related protein, the ectoadenosine diphosphatase (apyrase) of
chicken oviduct, and the protein was immunopurified
(19) . Here
we used a similar approach to generate monoclonal antibodies to the
chicken gizzard smooth muscle ecto-ATPase for the characterization and
purification of that enzyme. The monoclonal antibodies were screened
using an ATPase capture assay
(19) and characterized. Two known
proteins were immunoaffinity purified along with the ecto-ATPase,
suggesting a functional linkage. The distribution of this enzyme in
adult chicken tissues appears to be most concentrated in muscle
(cardiac, smooth, and skeletal) and brain. This distribution is
consistent with previous reports
(2, 6) ; however, the
apparent molecular weight of the recognized protein on Western blots is
different than reported previously
(2, 6) . Unlike those
previous reports, the Western blot results reported in this study agree
with the purification results
(8, 18) , establishing that
the 66-kDa glycoprotein is indeed the ecto-ATPase and that the avian
and mammalian enzymes are very similar in size and extent of
glycosylation.
The tissue distribution of
ecto-ATPase reported here identifies a 66-kDa protein found
predominantly in the excitable tissues, muscle (smooth, skeletal, and
cardiac) and brain. However, it must be noted that if there exist
isoforms of the ecto-ATPase that are differentially expressed in
different tissues, any tissue distribution data generated using
monoclonal antibodies may reflect the possibility that the single
epitope recognized by a given monoclonal antibody may not be present in
all isoforms (assuming there are tissue-specific isoforms). This
possibility is less likely in this case since we have used several
monoclonal antibodies and obtained similar tissue distributions, and
have been able to immunoaffinity purify the ecto-ATPase from several
tissues with the same monoclonal antibodies (not shown).
The tissue
distribution reported here is somewhat different than we previously
published using an anti-peptide antibody
(6) . One plausible
explanation for the different molecular masses reported for the
ecto-ATPase (approximately 66 versus 96 kDa) of the recognized
protein is that the anti-peptide antibody may have cross-reacted with
the more abundant protein, caldesmon. The ubiquitous protein,
caldesmon, contains a repeating sequence (EEE) found in the peptide
used previously as an antigen (NH
Prior to the results presented in this work,
there were no specific stimulators or probes available for use in
structural and functional studies of the ecto-ATPases. Although there
was a report that the ecto-ATPase from rat liver had been cloned and
sequenced
(37) , it seems likely that the Cell-CAM-105 adhesion
protein that was cloned is not the rat liver ecto-ATPase (contrary to
what was previously believed), since it can be partially separated
chromatographically from the Cell-CAM-105 adhesion protein.
The ecto-ATPase is a particularly difficult
protein to study because of its low abundance and susceptibility to
inactivation by many biological detergents, and therefore homologous or
more abundant proteins co-purifying with the ecto-ATPase are frequently
identified incorrectly as the
ecto-ATPase
(2, 3, 13) . Since the current data
utilizing the monoclonal antibodies agrees completely with both reports
of the purification of a muscle ecto-ATPase to homogeneity and very
high specific activity
(8, 18) , it is clear that the
66-kDa protein recognized by monoclonal antibodies described in this
work truly represents the ecto-ATPase and not some co-purifying,
related, or associated protein. Therefore, these monoclonal antibodies
will be powerful probes for the further investigation of the functions
of the ecto-ATPases.
++++,
very strong; +++, strong; ++, significant;
+, marginal; 0, no effect; ND, not determined; *, denotes
monoclonal antibodies that were further characterized.
This table is based on Figure 2. Western blot was
probed with monoclonal antibody 10 and quantitated by densitometry.
We thank Cleris Gil for providing the protein
sequencing data.
(
)
are
glycoproteins that exhibit divalent cation-dependent NTPase activity on
the extracellular side of the plasma membrane. These enzymes are
present in tissues in low abundance and have low substrate specificity
but have a very high specific
activity
(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The identities and functions of ecto-ATPases are the subject of a
recent review in which the nomenclature of ``E-type ATPases''
was proposed to describe these enzymes
(10) . The physiological
function of these enzymes is still unknown. However, several hypotheses
about putative ecto-ATPase function(s) have been proposed by
association and include 1) regulation of P
-purinergic
receptors, neurotransmission and signal transduction
(11) ; 2)
involvement in cellular adhesion and cancer
metastasis
(12, 13, 14, 15, 16) ;
3) platelet aggregation; and 4) regulation of NO release in smooth
muscle
(17) . Unfortunately, progress in the study of
ecto-ATPases has been greatly impeded as a result of the lack of a
specific inhibitor/probe and the purification difficulties associated
with the observed very low protein abundance in tissues.
Materials
Adult chicken tissues were obtained
fresh from a local slaughterhouse. All reagent grade chemicals and
buffer salts were purchased from Fisher or the Sigma. Protein
A-Sepharose 4B, digitonin, Nonidet P-40, CHAPS,
CE
, dimethyl pimelimidate dihydrochloride,
anti-mouse IgG-agarose, concanavalin A, and methyl
-D-mannopyranoside were purchased from Sigma.
Concanavalin A-Sepharose 4B was purchased from Pharmacia Biotech Inc.
Quantigold was purchased from Diversified Biotech. Enhanced
chemiluminescence (ECL) reagents were purchased from DuPont NEN.
Anti-mouse IgG-goat horseradish peroxidase conjugate was from
CalBiochem, and anti-rabbit IgG-goat horseradish peroxidase conjugate
was from Bio-Rad. CentriPrep 30, 50, and 100 concentrators and
Centricon 30 microconcentrators were from Amicon and Centrex UF-0.5 30
centrifugal ultrafilters were from Schleicher and Schuell. MemSep
quaternary ammonium anion exchange membrane-based columns were
purchased from Millipore-Waters. Immobilon-P
polyvinylidene fluoride membrane was from Millipore, and all
electrophoresis apparati, chemicals, and standards were from Bio-Rad.
Protein sequences were determined by the Protein Core Facility of the
Department of Pharmacology and Cell Biophysics at the University of
Cincinnati College of Medicine.
Membrane Preparation
Membranes were prepared from
whole tissue on a large scale (approximately 200 g) or small scale
(approximately 20 g), depending on the quantity of tissue available.
Tissues were handled in a cold room and were centrifuged at 4 °C.
Details of the large scale
(8) and small scale
(6) membrane preparations were described in those earlier
publications.
Protein Concentration
Protein concentration was
determined using the Bio-Rad dye binding technique, using the
modification of Stoscheck
(20) , and bovine serum albumin as the
standard. The Mg-ATPase activity was determined using a modification
(18) of the technique of Fiske and Subbarow
(21) . The
unstimulated ecto-Mg-ATPase specific activity of the gizzard microsomal
membranes was typically 200 µmol/mg/h.
Solubilization
For the experiments requiring
maintenance of ecto-ATPase activity, membrane-bound proteins were
solubilized with 1.0% digitonin at a protein concentration of 1 mg/ml
in 20 mM MOPS, 2 mM MgCl, pH 7.4 for 10
min at room temperature. The solubilized membranes were centrifuged at
48,000 rpm in a 50 Ti rotor (150,000
g) for 30 min to
pellet the remaining insoluble proteins. The supernatant containing the
solubilized proteins was removed and diluted accordingly.
Solubilization with digitonin preserved activity but was not very
efficient for extracting the ecto-ATPase, particularly with brain
membranes. Other detergents were tested for solubilization of total
protein from bovine brain membranes without concern for activity.
Detergents, including digitonin, CHAPS, Nonidet P-40,
n-dodecyl maltoside, and C
E
, were
evaluated at 0.5 and 1.0% with a membrane protein concentration of 1
mg/ml. Solubilization was performed as described above. The best
detergent for solubilization of total protein from brain tissue
membranes was determined to be 1.0% Nonidet P-40. Therefore 1.0%
Nonidet P-40 (1.0 mg/ml protein concentration) was used to solubilize
membrane proteins for all of the immunoaffinity purification
experiments, since maintenance of ATPase activity was not required.
Immunogen Preparation
Gizzard membranes (30 mg)
were solubilized twice with 1.0% digitonin (assumed 50% solubilization
of membrane proteins for the first solubilization) as described above.
The supernatants were pooled, diluted 5-fold with digitonin-free Buffer
A (10 mM MOPS, 2 mM MgCl, 0.1% digitonin,
pH 7.4) and incubated with 3-5 ml of concanavalin A-Sepharose 4B,
pre-equilibrated with Buffer A, for 1 h on ice. The concanavalin
A-Sepharose 4B was then poured into a column, washed 5 times with 5 ml
of Buffer A and 2 times with 5 ml of Buffer B (10 mM Tris-HCl,
2 mM MgCl
, 0.1% digitonin, pH 8.2). The
concanavalin A-Sepharose 4B column was connected to a quaternary
methylammonium anion MemSep column (size 1000), pre-equilibrated in
Buffer B, and eluted with 30 ml of 300 mM methyl
-D-mannopyranoside in Buffer B at 1 ml/min so that the
eluted glycoproteins would immediately bind to the anion-exchange
column for further purification. The quaternary methylammonium anion
MemSep column was washed with 10 ml of Buffer B and eluted with 7 ml of
50 mM NaCl in Buffer B at 1 ml/min, and 1-ml fractions were
collected. The peak of Mg-ATPase activity eluted in fractions
3-6. The peak of activity was pooled, concentrated, washed twice
with 1 ml of 250 mM Tris-HCl buffer, pH 6.8, and finally
concentrated to
200 µl in a Centricon-30 microconcentrator. The
sample was diluted 1:1 with 250 mM Tris-HCl buffer, pH 6.8,
saturated with sucrose and containing bromphenol blue as a tracking
dye. The sample was carefully loaded on a 6.0% acrylamide native gel
containing 0.1% digitonin and run at 180 V for 3 h; the glycine buffer
was replaced with fresh buffer after 90 min. The gel was developed
using a modification of the acid phosphatase zymogram of Nimmo and
Nimmo
(22) , resulting in the formation of a white calcium
phosphate precipitate where the ATP was hydrolyzed and thus detecting
the ecto-ATPase. (The calcium phosphate precipitate used to localize
the enzyme also acts as an adjuvant for antibody production.) The gel
band of activity was excised and stored at -20 °C. The gel
slices from three such immunogen purifications were pooled and washed 8
times in 25 mM Tris-HCl, 150 mM NaCl, pH 6.8, for 5
min/wash. This procedure washed away some of the calcium phosphate but
was needed to remove the majority of the digitonin, which is toxic when
injected into mice. The gel slices were then homogenized in 250 ml of
buffer using a Waring commercial blender at full speed for 1 min and
then centrifuged at 3,000 rpm in a JA-20 rotor for 10 min to pellet.
The pellet was resuspended in buffer to form a slurry of approximately
50% fragmented material, which was homogenized with three passes of a
Teflon homogenizer. The slurry, representing the final immunogen
product for injection, was divided into 1-ml aliquots and stored at
-20 °C.
Monoclonal Antibody Preparation, Evaluation, and
Isotyping
Four outbred Swiss mice were immunized with an
intraperitoneal injection of 1 ml of immunogen, day 1, and the
immunization protocol proceeded as described.(
)
After booster injection, the mouse spleenocytes were
harvested and fused with myeloma cells of strain NS1 as described
previously
(24) .
The hybridomas were grown in
microtiter dishes for about 2 weeks and subjected to the ATPase capture
assay using screening enzyme. ATPase capture assay of the monoclonal
tissue culture supernatants was performed as described
previously
(19) . The ATPase enzyme used to screen the monoclonal
antibodies in the ATPase capture assays consisted of the unpurified
supernatant from 30 mg of gizzard membranes solubilized twice with 1.0%
digitonin. 18 ATPase capture assay positive colonies were subcloned by
limiting dilution and were evaluated for their Western blot and
immunoprecipitation capabilities for ecto-ATPase (). Of
these 18 positives, five monoclonal antibody hybridomas were chosen to
further subclone, and larger quantities of antibody were produced in
tissue culture. The monoclonal antibodies from these five overgrown
supernatants were isotyped using the Sigma immunotype kit (ISO-1).
Immunoprecipitation
5 mg of chicken gizzard
membranes at 1 mg/ml were solubilized with 1.0% digitonin in 20
mM MOPS, 2 mM MgCl, pH 7.4. After
solubilizing for 10 min with stirring at room temperature, the soluble
proteins were separated from the insoluble proteins by centrifugation
at 48,000 rpm (150,000
g) in a 50 Ti rotor at 4 °C
for 30 min. Aliquots of the supernatant (200 µl) were diluted 1:5
with TBS and incubated with the appropriate monoclonal antibody (250
µl of tissue culture supernatant) in the presence of protein
G-agarose at 4 °C overnight on a Labquake Shaker. Controls
containing no protein G-agarose, no antibody, or no protein G-agarose
and no antibody were run in parallel. Immunoprecipitation of
ecto-ATPase was evaluated by assaying the beads and supernatants for
ecto-Mg-ATPase activity as described above.
Monoclonal Antibody Purification and Immunoaffinity
Column Production
Monoclonal antibodies in tissue culture fluid
were incubated with 1 ml of anti-mouse IgG-agarose, preequilibrated in
TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5), overnight
at 4 °C using a Labquake Shaker. The anti-mouse
IgG-agarose/monoclonal antibody was poured into a column and washed
with TBS. The anti-mouse/monoclonal column was either cross-linked to
form an anti-mouse-agarose-monoclonal column (method described below)
or the purified monoclonal antibodies were eluted from the column with
200 mM glycine buffer, pH 3.0 at 1.0 ml/min. The 1-ml
fractions were immediately neutralized with Tris base, and protein was
quantitated by optical density at 280 nm. Fractions containing protein
were pooled and incubated with 0.4 ml of protein A-Sepharose 4B
overnight at 4 °C using a Labquake Shaker. The protein A-Sepharose
4B/monoclonal was poured into a column and washed 3 times with 1.5 ml
of 200 mM sodium borate buffer, pH 9.0. The protein
A-Sepharose 4B/monoclonal column (or anti-mouse-agarose/monoclonal
column) was adjusted to a final volume of 4.0 ml with 200 mM
sodium borate buffer, pH 9.0, and the cross-linker, dimethyl
pimelimidate, was added to a final concentration of 20 mM.
After 30 min at room temperature on a rocking platform, the
cross-linking reaction was stopped by washing the column with, and
incubating in, 200 mM ethanolamine, pH 8.0 for 2 h at room
temperature. The column was then washed extensively with TBS and stored
in TBS containing 0.02% NaN at 4 °C.
Immunoaffinity Chromatography
Chicken gizzard
membranes were solubilized at 1.0 mg/ml with 1.0% Nonidet P-40 as
described above. The supernatants were diluted 5-10-fold with TBS
to reduce the detergent concentration to 0.2% prior to incubation with
the bead-bound monoclonal antibody (the antibody had been cross-linked
to either protein A-Sepharose or anti-mouse IgG-agarose) overnight on a
platform rocker at 4 °C. The monoclonal antibody/sample was poured
into a column and washed with 10 ml of TBS containing 0.1% Nonidet
P-40. The approximately 0.5-ml column was eluted with 10 ml of 200
mM glycine buffer, 0.1% Nonidet P-40, 0.05% NaN,
pH 3.0, at 1 ml/min, and the 1-ml fractions were neutralized
immediately with Tris base. The protein concentration of each fraction
was determined by colloidal gold
(25) .
Chemical Deglycosylation
Chemical deglycosylation
of chicken gizzard ecto-ATPase was done according to Horvath et
al.(26) , after precipitation of 150 µg of chicken
gizzard membranes with 4 volumes of -20 °C acetone for 30 min
at -20 °C.
Electrophoresis and Western Blot Analysis
SDS-PAGE
was performed according to Laemmli
(27) . Samples were boiled for
5 min in reducing SDS sample buffer (20 mM dithiothreitol)
with 8 M urea (or without urea for protein sequencing
purposes). Gels were either 0.75 mm (analytical) or 1.5 mm (native)
thick. The native gel consisted of a 6% Laemmli resolving gel
containing 0.1% digitonin and no SDS as described
previously
(28) . The gels were either stained with silver
according to Ansorge
(29) or electroblotted onto 0.2-µm
polyvinylidene fluoride membranes for 2 h at 33 V in 10 mM
CAPS buffer, pH 11.0
(30) . For sequencing, the blots were
stained with Coomassie Brilliant Blue for 1 min and destained, and the
bands were excised. For Western blot analysis, the unstained membranes
were blocked with 5% nonfat dry milk in TBS for 1 h at room
temperature. The primary antibody was diluted into TBS containing 5%
milk and 0.02% NaN and incubated with the blot overnight.
After washing and incubation for 1 h with an anti-mouse horseradish
peroxidase secondary antibody (containing no NaN
),
immunoreactivity was detected by chemiluminescence with the DuPont NEN
ECL reagents as described by the manufacturer.
RESULTS
Monoclonal antibodies were generated against a preparation of
digitonin solubilized, partially purified, native ecto-ATPase isolated
from chicken gizzard smooth muscle membranes. The 18 monoclonal
antibodies were evaluated for their ability to recognize the chicken
gizzard ecto-ATPase by Western analysis, immunoprecipitation of
ecto-ATPase activity, and stimulation of ecto-ATPase activity
(). Based on the results presented in , five
monoclonal antibodies (6, 10, 12, 15, and 16) were chosen for further
subcloning and large scale production. As determined by Western blot
analysis of chicken gizzard membranes (see Fig. 1), monoclonal
antibodies 6, 10, and 15 all recognized a glycoprotein with a molecular
size of approximately 66 kDa, which has a core protein molecular mass
of 53 kDa, consistent with the apparent molecular masses observed for
both the purified chicken gizzard ecto-ATPase
(8) and rabbit
skeletal muscle t-tubule ecto-ATPase
(18) . However, monoclonal
antibodies 12 and 16 recognized proteins on Western blots with apparent
molecular masses of approximately 160 and 90 kDa, respectively (data
not shown). The largest stimulation of ecto-ATPase activity
(approximately 13-fold) was observed with monoclonal antibody 6. This
stimulation is very reminiscent in both manner and magnitude to the
approximately 19-fold maximal stimulation previously observed with
concanavalin A
(2, 8) and may be indicative of an
oligomerization-induced activation, as was proposed for the lectin
stimulation mechanism
(8) .
Figure 1:
Detection of
intact and deglycosylated chicken gizzard ecto-ATPase with monoclonal
antibodies. Chicken gizzard membranes (15 µg/well) were
analyzed for the presence of ecto-ATPase by Western analysis with
monoclonal antibodies 6, 10, and 15 before (-) and after (+)
chemical deglycosylation with trifluoromethanesulfonic acid
(TFMSA) as described under ``Experimental
Procedures.'' The migration position of the intact and fully
deglycosylated proteins are indicated at 66 and 53 kDa,
respectively.
The distribution of the 66-kDa
ecto-ATPase in adult chicken tissues was analyzed using monoclonal
antibody 10 (which was experimentally determined to give the best
Western blot detection of the 66-kDa ecto-ATPase from chicken tissues
other than gizzard, data not shown). Western blots of adult chicken
tissues with this antibody revealed the expected 66-kDa ecto-ATPase
immunoreactive band (Fig. 2). The protein abundance of the
ecto-ATPase was highest in gizzard (smooth muscle), followed by brain
(excluding cerebellum), stomach, skeletal muscle (breast), and heart,
as shown in Fig. 2and . Significant amounts of
ecto-ATPase were also found in pancreas and intestine. The relatively
high abundance of ecto-ATPase in those tissues could be due to the
presence of smooth muscle membranes in those tissue preparations.
Figure 2:
Distribution of ecto-ATPase in adult
chicken tissues. 10 µg of membranes from adult chicken tissues were
resolved by SDS-PAGE and Western blotted with monoclonal antibody 10.
The ecto-ATPase is detected as a broad immunoreactive band at 66
kDa.
The high specificity of all five monoclonal antibodies was exploited
to purify the ecto-ATPase from solubilized membranes. After
solubilization of gizzard membranes with Nonidet P-40 and subsequent
dilution with TBS, samples were immunopurified with each monoclonal
antibody affinity column. SDS-PAGE/silver stain analysis of the
concentrated eluants from these columns revealed that all five
antibodies immunopurified the ecto-ATPase as evidenced by the presence
of a 66 kDa band in all samples (Fig. 3). The intensity of the
faint band observed near 130 kDa in Fig. 3was decreased upon
addition of fresh reductant to the SDS sample buffer prior to heating
and electrophoresis, an indication that this band is a disulfide dimer
of the 66-kDa ecto-ATPase (not shown). Also, immunopurified bands at
90 and 160 kDa were observed with monoclonal antibodies 16 and 12,
respectively, consistent with the molecular masses of the proteins
recognized by these antibodies on Western blots. The immunopurified
protein band at 66 kDa was sequenced and found to be identical to the
N-terminal sequence of chicken gizzard ecto-ATPase
(8) . This
sequence data further verified that the antibodies were specific for
ecto-ATPase and allowed a longer (25 amino acid, see I)
N-terminal sequence to be determined than had been published previously
(12 amino acids,
(8) ). The co-immunoaffinity purified protein
bands at approximately 90 and 160 kDa were also sequenced and found to
be known proteins (I).
Figure 3:
SDS-PAGE
analysis of immunoaffinity chromatography purified chicken gizzard
ecto-ATPase. Chicken gizzard membranes were solubilized and subjected
to immunoaffinity chromatography as described under ``Experimental
Procedures.'' An aliquot of each column's eluant was acetone
precipitated and analyzed by SDS-PAGE followed by silver staining (29).
Lane1, Bio-Rad high molecular mass standards (0.2
µg); lane2, Bio-Rad low molecular mass standards
(0.2 µg); lanes3-7, immunoaffinity
purified ecto-ATPase using monoclonal antibodies 6, 10, 12, 15, and 16,
respectively. Electrophoretic migration positions of ecto-ATPase (66
kDa), LEP100 (90 kDa), integrin (
160 kDa), and the ecto-ATPase
dimer (doublearrow at
130 kDa) are indicated in
the right-hand margin.
DISCUSSION
Immunoaffinity chromatography using all 5 of the monoclonal
antibodies chosen for further study (all of which immunoprecipitated
ecto-ATPase activity, see ) resulted in the isolation of
the 66-kDa ecto-ATPase (see Fig. 3) expected from previous
purification work
(8, 18) . An extended N terminus of 25
amino acids was obtained for the ecto-ATPase, a sequence that showed
significant sequence homology with the N terminus of the mouse
lysosomal-associated membrane protein (I, LAMP-1). Three
of the five antibodies recognized the 66-kDa ecto-ATPase on Western
blots. The other two antibodies recognized proteins of approximately 90
and 160 kDa. These ``associated'' proteins were purified by
immunoaffinity chromatography followed by SDS-PAGE and N-terminal
protein sequencing. (Of course, it is possible that these
``associated'' proteins are not associated with the
ecto-ATPase but instead share a common epitope with the ecto-ATPase,
which is recognized by the same monoclonal antibody. If this is the
case, then this is still an important finding, since such shared
epitopes may also suggest structural and functional relatedness of the
ecto-ATPase with these co-immunopurified proteins.) The approximately
90-kDa protein was determined to be identical to LEP100, a glycoprotein
that shuttles between the lysosomal, endosomal, and plasma membranes
(31-33). It seems reasonable that this is the same approximately
90-kDa protein that was found to co-purify with the chicken gizzard
ecto-ATPase in ``conventional'' chromatography (see
Fig. 2
in Ref. 8). The 160-kDa protein was also purified and
sequenced and found to be identical to integrin
(34) by
N-terminal sequence analysis (see I). Integrin is a matrix
glycoprotein important in the recognition and adhesions of cells to
other cells and to the extracellular matrix (34-36). These
findings of sequence homologies with known proteins seem to be
consistent with the theory that the ecto-ATPase may be involved with
membrane/membrane recognition and/or
adhesion
(2, 13, 37) . However, consistent with
our previous findings
(7, 8) , we have not detected
either T-cadherin
(2) or N-CAM
(13) co-immunoaffinity
purifying with the ecto-ATPase.
-KILSGEEEGVFG). Caldesmon
has both a low and high molecular weight form and is abundant in
gizzard smooth muscle where it is proposed to function as a regulator
of actin filaments
(38) . The low molecular weight form of
caldesmon has been observed to migrate anomalously on
SDS-PAGE
(38) , and so an apparent molecular mass for the smaller
form of 96 kDa is not unreasonable. In our previous work
(6) ,
the gizzard was observed to have developmentally regulated 96 and 130
kDa immunoreactive bands, consistent with what is known about the
regulation of the low and high molecular weight forms of caldesmon in
gizzard. Subsequent unpublished experiments performed in our laboratory
showed that the immunoreactivity to the anti-peptide antibody was
extracted from the gizzard membranes by boiling, again consistent with
the known properties of caldesmon
(39) , and inconsistent with
the properties of an integral membrane protein like the ecto-ATPase.
Nonetheless, the apparent molecular mass (96 kDa in most tissues, 96
and 130 kDa seen in gizzard tissue) and tissue distribution of the
ecto-ATPase previously found using the anti-peptide antibody was very
similar to that published by Cunningham et al.(2) using an antibody that inhibited ecto-ATPase activity.
Therefore, it seems likely that the protein detected on Western blots
in both of these previous studies was the more abundant caldesmon
protein rather than the ecto-ATPase. If the conclusions stated above
are correct, then it seems likely that at least a subpopulation of the
caldesmon protein may be associated with the ecto-ATPase, since an
anti-peptide antibody reactive protein near 96 kDa (presumably
caldesmon) co-eluted with the peak of ecto-ATPase activity in chicken
brain
(7) , and an antibody that (from the arguments given above)
apparently recognizes caldesmon on Western blots inhibited ATPase
activity
(2) .
(
)
Therefore, the protein that was cloned and sequenced and
used as an antigen for the production of antibodies was not the
ecto-ATPase, but instead it was the co-purifying Cell-CAM-105 adhesion
molecule
(40) . By analogy with both the rabbit skeletal muscle
and the chicken smooth muscle ecto-ATPases that have been purified to
homogeneity in our laboratory (which are both approximately 70-kDa
proteins
(8, 18) ), the rat liver ecto-ATPase is probably
the approximately 70-kDa protein first identified as the
ecto-ATPase
(41) .
Table:
Characterization of monoclonal antibodies raised
against the chicken gizzard ecto-ATPase
Table:
Tissue distribution of 66-kDa ecto-ATPase in
adult chicken
Table:
N-terminal sequences of immunoaffinity
chromatography-purified chicken gizzard proteins; homologies with known
proteins
E
,
polyoxyethylene-9-lauryl ether.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.