Chicken oviductal ecto-ATP-diphosphohydrolase. Purification and characterization.

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 micronol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.

Identification and immunolocalization of ecto-ATPDase in chicken stomach.

The distribution of the extracellular adenosine tri- and di-phosphatase (ecto-ATPDase) in adult chicken tissues was investigated using a monoclonal antibody (MC18) generated previously from chicken oviduct. Ecto-ATPDase was determined to be most abundant in stomach by Western blot analysis of crude tissue homogenates. The ecto-ATPDase activity from solubilized stomach microsomes and a purified oviduct control was depleted 64% and 72%, respectively, by immunoprecipitation with MC18. Both oviduct and stomach ecto-ATPDases had an M(r) of approximately 80 kDa based on SDS-PAGE analysis. In addition, the enzymology of the ecto-ATPDase from both tissues was very similar. It is concluded that the same ecto-ATPDase is present in stomach and oviduct. Furthermore, immunolocalization of the stomach ecto-ATPDase with MC18 showed the enzyme to be localized in the apical membranes of the oxyntico-peptic cells, suggesting a role for the ecto-ATPDase in secretion.

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.

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.