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Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside
Bernd U Failer1, Norbert Braun, Herbert Zimmermann
1Arbeitskreis Neurochemie, Biozentrum der J. W. Goethe-Universitaet, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany.
The Journal of Biological Chemistry
|August 9, 2002
Summary
We identified a calcium-dependent nucleoside diphosphatase in rat brain. This enzyme, located in the endoplasmic reticulum, may play a role in protein quality control during glycosylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Nucleoside diphosphatases are enzymes involved in nucleotide metabolism.
- Secreted apyrases in insects hydrolyze ATP and ADP.
- The function of Ca(2+)-dependent nucleoside diphosphatases in eukaryotes is not fully understood.
Purpose of the Study:
- To isolate and characterize the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase from rat brain.
- To investigate the enzyme's localization, substrate specificity, and kinetic properties.
- To explore the potential role of this enzyme in cellular processes.
Main Methods:
- cDNA isolation and sequencing.
- mRNA expression analysis using Northern blotting.
- Immunohistochemical analysis of recombinant protein localization.
- Enzyme activity assays to determine substrate specificity and kinetics.
Main Results:
- Isolated cDNA encodes a 403-amino acid protein (45.7 kDa) with high similarity to insect apyrases.
- mRNA expressed in all rat tissues, with two major transcripts.
- Recombinant enzyme localized to the endoplasmic reticulum and pre-Golgi intermediates.
- Enzyme efficiently hydrolyzes UDP, GDP, and IDP, with strong Ca(2+) activation and a K(m) for UDP of 216 µM.
- No significant hydrolysis of ADP, nucleoside triphosphates, or monophosphates observed.
Conclusions:
- Rat brain Ca(2+)-dependent nucleoside diphosphatase is an ER-associated membrane protein.
- The enzyme's substrate specificity suggests a role in UDP-dependent glycosylation pathways.
- Potential involvement in endoplasmic reticulum-associated quality control mechanisms.