Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

Candy A. Heyen, Vincent S. Tagliabracci, Lanmin Zhai, Peter J. Roach

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

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 [β-32P]UDP-glucose to [32P]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 Mg2+ and was greatest at alkaline pH above 8. Kinetic analysis indicated a Km of ∼4 mM for UDP-glucose and ∼0.3 mM for ADP-ribose. Based on Vmax/Km values, the enzyme was ∼20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of Mr 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.

Original languageEnglish (US)
Pages (from-to)1414-1418
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume390
Issue number4
DOIs
StatePublished - Dec 25 2009

Keywords

  • ADP-ribose
  • Glycogen
  • Nudix hydrolase
  • Nudt14
  • Pyrophosphatase
  • UDP-glucose

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

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