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.
Strobel
R S
RS
Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.
Nagy
A K
AK
Knowles
A F
AF
Buegel
J
J
Rosenberg
M D
MD
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002645:Chickens; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010057:Oviducts / Q000201:enzymology*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D000251:Adenosine Triphosphatases; C027201:ectoATPase