Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase.
We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) 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. Ca(2+)-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 Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum.
Failer
Bernd U
BU
Arbeitskreis Neurochemie, Biozentrum der J. W. Goethe-Universitaet, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany.
Braun
Norbert
N
Zimmermann
Herbert
H
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D016466:CHO Cells; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D017766:Acid Anhydride Hydrolases; C023837:nucleoside-diphosphatase; D002118:Calcium