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J. Biol. Chem., Vol. 277, Issue 40, 36978-36986, October 4, 2002
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From the Arbeitskreis Neurochemie, Biozentrum der J. W. Goethe-Universitaet, Marie-Curie-Strasse 9, D-60439 Frankfurt am
Main, Germany
We have isolated and characterized the cDNA
encoding a Ca2+-dependent nucleoside
diphosphatase (EC 3.6.1.6) related to two secreted ATP- and
ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex
lectularius and Phlebotomus papatasi. The rat
brain-derived cDNA has an open reading frame of 1209 bp encoding a
protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated,
revealing two major transcripts with varying preponderance. The
immunohistochemical analysis of the Myc-His-tagged enzyme expressed in
Chinese hamster ovary cells revealed its association with the
endoplasmic reticulum and also with pre-Golgi intermediates.
Ca2+-dependent nucleoside diphosphatase is a
membrane protein with its catalytic site facing the organelle lumen.
It hydrolyzes nucleoside 5'-diphosphates in the order UDP
>GDP = IDP >>>CDP but not ADP. Nucleoside 5'-triphosphates
were hydrolyzed to a minor extent, and no hydrolysis of nucleoside
5'-monophosphates was observed. The enzyme was strongly activated by
Ca2+, insensitive to Mg2+, and had a
Km for UDP of 216 µM.
Ca2+-dependent nucleoside diphosphatase
may support glycosylation reactions related to quality control in the
endoplasmic reticulum.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AJ 312208 and AJ 312207.
Cloning, Expression, and Functional Characterization of a
Ca2+-dependent Endoplasmic Reticulum Nucleoside
Diphosphatase*
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB 269, A4) and the Fonds der Chemischen
Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-69-798-29602;
Fax: 49-69-798-29606; E-mail:
h.zimmermann@zoology.uni-frankfurt.de.
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