Properties of and proteins associated with the extracellular ATPase of chicken gizzard smooth muscle. A monoclonal antibody study.
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.
Stout
J G
JG
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575, USA.
Strobel
R S
RS
Kirley
T L
TL
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD; D030161:Avian Proteins; D015153:Blotting, Western; D002645:Chickens / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D005895:Gizzard, Avian / Q000201:enzymology*; D007163:Immunosorbent Techniques; D016023:Integrins / Q000737:chemistry / Q000302:isolation & purification; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology*; D009928:Organ Specificity
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D030161:Avian Proteins; D016023:Integrins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009124:Muscle Proteins; C066541:LEP100 protein, Gallus gallus; D000251:Adenosine Triphosphatases; C027201:ectoATPase