Properties of and Proteins Associated with the Extracellular ATPase of Chicken Gizzard Smooth Muscle

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.

OppA, the Substrate-Binding Subunit of the Oligopeptide Permease, Is the Major Ecto-ATPase ofMycoplasma hominis

“…Thus, binding of ATP appears to be sufficient to cause protection against trypsin digestion whereas mutation of the whole Walker A motif is required to decrease ATP binding and hydrolysis. ecto-kinase substrate concentration (33), (iii) involvement in signal transduction (2,25,27), and (iv) involvement in cellular adhesion (17,20,42). So far the physiological role of OppA in Mycoplasma hominis remains speculative.…”

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

“…Ecto-nucleotidases have now been characterized from a diversity of tissues and species, ranging from insects to parasites to plants and to mammals (6,10,24,25). Substrate specificity of ATPDases varies broadly as individual systems are studied.…”

Immunolocalization of the Ecto-ATPase and Ecto-apyrase in Chicken Gizzard and Stomach

“…We are interested in determining structural and functional similarities and differences between the ecto-ATPases (which do not hydrolyze nucleoside diphosphates) and ecto-apyrases (ectoATPDases), which hydrolyze nucleoside diphosphates at similar rates as nucleoside triphosphates. Previously (17,18), we purified and characterized the ecto-ATPase from chicken smooth muscle (gizzard). In addition, we showed that chicken stomach was a good source of ecto-ATPDase (19) that was immunologically indistinguishable from that purified by others from chicken oviduct and liver (20).…”