Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.
GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.
Ning
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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Elbein
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D006005:Phosphorylases; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; C418972:glucose-1-phosphate guanylyltransferase; D008274:Magnesium