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) i s 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 e valuated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclon al antibodies identified by an ATPase capture assay were tested for ef fects on ATPase activity as well as for their Western blot and immunop recipitation 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 o f 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 analys is 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 recognize d proteins of approximately 90 and 160 kDa on Western blots. The 90-kD a co-immunopurifying (and presumably associated or re lated) protein w as identified by N-terminal analysis as LEP100, a glycoprotein that sh uttles between the plasma and lysosomal membranes. The approximately 1 60-kDa co-immunopurifying protein was identified by N-terminal analysi s as integrin, a protein involved in extracellular contacts with adhes ion molecules, Extended N-terminal sequence analysis of the immunopuri fied 66-kDa ecto-ATPase revealed some sequence homology with mouse lys osomal associated membrane protein. Tissue distribution of the ecto-AT Pase showed that the highest levels of protein were expressed in muscl e tissues (cardiac, skeletal, and smooth) and brain.