An enzyme-coupled continuous spectrophotometric assay for sugar nucleotide dependent glycosyltransferases.

WAYLLACE, N. Z.; VALDEZ, H.; MERAS, A.; UGALDE, R.; BUSI, M. V.; DIEGO FABIAN GOMEZ CASATI

MOLECULAR BIOLOGY REPORTS

SPRINGER

Lugar: Berlin; Año: 2012 vol. 39 p. 585 - 591

0301-4851

Abstract The metabolic pathways leading to the syn- thesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the a-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 Esche- richia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.