Cloning, expression, and functional characterization of a Ca2+-dependent endoplasmic reticulum nucleoside diphosphatase

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 K-m for UDP of 216 muM. Ca2+-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum.