NDLI: An enzyme-coupled continuous spectrophotometric assay for glycogen synthases

Content Provider	Springer Nature Link
Author	Wayllace, Nahuel Z. Valdez, Hugo A. Merás, Andrea Ugalde, Rodolfo A. Busi, Maria V. Gomez Casati, Diego F.
Copyright Year	2011
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.
Starting Page	585
Ending Page	591
Page Count	7
File Format	PDF
ISSN	03014851
Journal	Molecular Biology Reports
Volume Number	39
Issue Number	1
e-ISSN	15734978
Language	English
Publisher	Springer Netherlands
Publisher Date	2011-05-17
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Glycosyltransferase Glycogen synthase Agrobacterium tumefaciens Animal Biochemistry Animal Anatomy / Morphology / Histology
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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