TAILIEUCHUNG - Báo cáo khoa học: Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Saccharomyces cerevisiaepossesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a con-served tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced inEscherichia coliand purified. By tryptic diges-tion and reversed phase chromatography a peptide (residues 219–246 of the complete Glg2p sequence) was isolated that contained 4–25 glucosyl residues | Eur. J. Biochem. 271 3978-3989 2004 FEBS 2004 doi Yeast glycogenin Glg2p produced in Escherichia coh is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation Tanja Albrecht1 Sophie Haebel2 Anke Koch1 Ulrike Krause1 Nora Eckermann1 and Martin Steup1 Institute of Biochemistry and Biology and interdisciplinary Center for Mass Spectrometry of Biopolymers University of Potsdam Potsdam-Golm Germany Saccharomyces cerevisiae possesses two glycogenin isoforms designated as Glg1p and Glg2p that both contain a conserved tyrosine residue Tyr232. However Glg2p possesses an additional tyrosine residue Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p carrying a C-terminal His6 tag was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide residues 219-246 of the complete Glg2p sequence was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30 less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially .

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