©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Properties of and Proteins Associated with the Extracellular ATPase of Chicken Gizzard Smooth Muscle
A MONOCLONAL ANTIBODY STUDY (*)

James G. Stout (1), Randy S. Strobel (2), Terence L. Kirley (1)(§)

From the (1) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575 and the (2) Department of Biology, Saint John's University, Collegeville, Minnesota 56321

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Extracellular ATPases (ecto-ATPases)() 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.

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.


EXPERIMENTAL PROCEDURES

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 CE, 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.

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-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) .

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.() 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) .

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.

  
Table: Characterization of monoclonal antibodies raised against the chicken gizzard ecto-ATPase

++++, very strong; +++, strong; ++, significant; +, marginal; 0, no effect; ND, not determined; *, denotes monoclonal antibodies that were further characterized.


  
Table: Tissue distribution of 66-kDa ecto-ATPase in adult chicken

This table is based on Figure 2. Western blot was probed with monoclonal antibody 10 and quantitated by densitometry.


  
Table: N-terminal sequences of immunoaffinity chromatography-purified chicken gizzard proteins; homologies with known proteins



FOOTNOTES

*
This work was supported by Grants RO1 AR38576 (to T. L. K.), K04 AR01841 (to T. L. K.), and T32-HL07382 (to J. G. S.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0575. Tel.: 513-558-2353; Fax: 513-558-1169; E-mail: kirleytl@ucbeh.san.uc.edu.

The abbreviations used are: ecto-ATPase, extracellular adenosine triphosphatase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MOPS, 3-(N-morpholino)propanesulfonic acid; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CE, polyoxyethylene-9-lauryl ether.

R. S. Strobel, A. K. Nagy, J. Buegel, A. F. Knowles, and M. D. Rosenberg, submitted for publication.

T. L. Kirley and S.-H. Lin, unpublished results.


ACKNOWLEDGEMENTS

We thank Cleris Gil for providing the protein sequencing data.


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