title: An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.
abstract: The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina.
authors.forename: Nahuel Z
authors.initials: NZ
authors.lastname: Wayllace
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Valdez
authors.forename: Andrea
authors.initials: A
authors.lastname: Merás
authors.forename: Rodolfo A
authors.initials: RA
authors.lastname: Ugalde
authors.forename: Maria V
authors.initials: MV
authors.lastname: Busi
authors.forename: Diego F
authors.initials: DF
authors.lastname: Gomez-Casati
mesh_terms: D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006003:Glycogen / Q000096:biosynthesis*; D006006:Glycogen Synthase / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D013053:Spectrophotometry / Q000379:methods*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D017931:DNA Primers; D000245:Adenosine Diphosphate Glucose; D006003:Glycogen; D006006:Glycogen Synthase; D014532:Uridine Diphosphate Glucose