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Originally published In Press as doi:10.1074/jbc.M201656200 on August 6, 2002

J. Biol. Chem., Vol. 277, Issue 40, 36978-36986, October 4, 2002
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Cloning, Expression, and Functional Characterization of a Ca2+-dependent Endoplasmic Reticulum Nucleoside Diphosphatase*

Bernd U. Failer, Norbert Braun, and Herbert ZimmermannDagger

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.


* 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.

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.

Dagger To whom correspondence should be addressed. Tel.: 49-69-798-29602; Fax: 49-69-798-29606; E-mail: h.zimmermann@zoology.uni-frankfurt.de.


Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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