Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene.
Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [beta-(32)P]UDP-glucose to [(32)P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg(2+) and was greatest at alkaline pH above 8. Kinetic analysis indicated a K(m) of approximately 4mM for UDP-glucose and approximately 0.3mM for ADP-ribose. Based on V(max)/K(m) values, the enzyme was approximately 20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M(r) 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.
Heyen
Candy A
CA
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Tagliabracci
Vincent S
VS
Zhai
Lanmin
L
Roach
Peter J
PJ
D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D005958:Glucosephosphates / Q000378:metabolism; D051379:Mice; D010802:Phylogeny; D055503:Protein Multimerization; D011755:Pyrophosphatases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D000096964:Nudix Hydrolases
D005958:Glucosephosphates; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D014532:Uridine Diphosphate Glucose; D000096964:Nudix Hydrolases; C031590:glucose-1-phosphate; C023255:UDPglucose pyrophosphatase; C546204:Nudt14 protein, mouse