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Chicken oviductal ecto-ATP-diphosphohydrolase. Purification and characterization
R S Strobel1, A K Nagy, A F Knowles
1Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.
The Journal of Biological Chemistry
|July 5, 1996
Summary
Researchers purified a chicken oviduct ecto-ATP diphosphohydrolase (ATPDase) to homogeneity, achieving over 25,000-fold purification. This enzyme exhibits the highest specific activity reported for an ATPDase, offering insights into ecto-ATPase functions.
Area of Science:
- Biochemistry
- Enzymology
- Cell Biology
Background:
- Ecto-ATP diphosphohydrolases (ATPDases) play crucial roles in cellular signaling and extracellular purinergic pathways.
- Vesiculosomes shed from chicken oviduct are a potential source of novel ectoenzymes.
Purpose of the Study:
- To purify and characterize an ecto-ATPDase from chicken oviduct vesiculosomes.
- To determine the enzymatic properties and molecular mass of the purified enzyme.
Main Methods:
- Purification involved filtration, differential centrifugation, exclusion chromatography, Nonidet P-40 extraction, ion exchange chromatography, lentil-lectin-Sepharose 4B chromatography, and immunoaffinity chromatography.
- Enzyme activity was measured using MgATP as a substrate.
- Molecular mass was determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis.
Main Results:
- Achieved over 25,000-fold purification of the ecto-ATPDase.
- The purified enzyme displayed a specific activity >800 µmol/min/mg protein with MgATP, the highest reported for an ATPDase.
- The enzyme hydrolyzed other nucleoside triphosphates and diphosphates at varying rates.
- Determined the molecular mass of the purified glycoprotein to be 80 kDa.
Conclusions:
- Successfully purified a highly active ecto-ATPDase from chicken oviduct vesiculosomes.
- The characterized enzyme provides a valuable model for studying ecto-ATPDase function and regulation.
- Further research can explore the specific physiological roles of this enzyme in the oviduct.