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Expression and characterization of human ecto-ATPase and chimeras with CD39 ecto-apyrase
1Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575, USA.
IUBMB Life
|November 22, 2000
Summary
Human ecto-ATPase (eNTPDase2) shows unique enzyme activity, stabilized by concanavalin A and disuccinimidyl suberate. Its N-terminal region dictates nucleotide specificity, while the C-terminal region is involved in cross-linking-induced activity enhancement.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human ecto-ATPase (eNTPDase2/CD39L1) belongs to the ecto-nucleoside triphosphate diphosphohydrolase family.
- This enzyme class plays roles in various physiological processes by hydrolyzing extracellular nucleotides.
- Understanding eNTPDase2's specific enzymatic properties is crucial for elucidating its biological functions.
Purpose of the Study:
- To express and characterize human ecto-ATPase (eNTPDase2) in COS cells.
- To investigate the unusual enzymology of eNTPDase2, including its substrate specificity and regulation.
- To determine the role of the N-terminal and C-terminal regions in eNTPDase2 function using chimeric constructs with CD39 (eNTPDasel).
Main Methods:
- Expression and characterization of human ecto-ATPase (eNTPDase2) in COS cells.
- Enzyme kinetic assays to study ATP hydrolysis and substrate specificity.
- Construction and analysis of chimeric proteins between eNTPDase2 and CD39 (eNTPDasel).
- Chemical cross-linking studies using disuccinimidyl suberate.
Main Results:
- Human ecto-ATPase (eNTPDase2) exhibited nonlinear hydrolysis of ATP over time.
- Enzyme activity was enhanced and stabilized by concanavalin A and disuccinimidyl suberate.
- The N-terminal half of eNTPDase2 was found to be critical for determining relative nucleoside di- and triphosphatase activities.
- Disuccinimidyl suberate cross-linking suggested interaction with the C-terminal half, increasing activity in both chimeric constructs.
Conclusions:
- Human ecto-ATPase (eNTPDase2) possesses distinct enzymatic properties compared to other eNTPDase family members.
- Glycosylation and oligomerization (tetramer) are important for eNTPDase2 activity, consistent with the family.
- The N-terminal region plays a key role in substrate specificity, while the C-terminal region is implicated in regulatory cross-linking.
- These findings provide insights into the structure-function relationships of the eNTPDase family.