CHICKEN OVIDUCTAL ECTO-ATP-DIPHOSPHOHYDROLASE - PURIFICATION AND CHARACTERIZATION

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity f rom vesiculosomes shed from chicken oviduct, First, the ecto-ATPDase-e nriched vesiculosomes were concentrated by filtration, differential ce ntrifugation, and exclusion chromatography, Next, the nonionic deterge nt, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculoso mal membranes, and the solubilized enzyme was further purified by ion by ion exchange (DEAE-Bio-Gel) and lentil lectin-Sepharose 4B chromato graphy, In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase, More than 25,000-fold purification w as achieved, Specific activity of the purified enzyme was greater than 800 mu mol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase, The enzyme also hydrolyzed other nucleo side triphosphates in the presence of magnesium at similar rates and C aATP and MgADP at lower rates, The molecular mass of the purified glyc oprotein was 80 kDa as deter mined by SDS-polyacrylamide gel electroph oresis and Western blot analysis, Based on its enzymatic properties, t he relationship of the chicken oviduct ecto-ATPDase with other reporte d ATPDases and ecto-ATPases is discussed.