An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.

WAYLLACE, N.Z.; VALDEZ, H.A.; MERAS, A.; UGALDE, R.A.; BUSI, M.V.; GOMEZ-CASATI, D.F

MOLECULAR BIOLOGY REPORTS

SPRINGER

Año: 2012 vol. 39 p. 585 - 591

0301-4851

The metabolic pathways leading to the synthesisof bacterial glycogen involve the action of severalenzymes, among which glycogen synthase (GS) catalyzesthe elongation of the a-1,4-glucan. GS from Agrobacteriumtumefaciens uses preferentially ADPGlc, although UDPGlccan also be used as glycosyl donor with less efficiency. Wepresent here a continuous spectrophotometric assay for thedetermination of GS activity using ADP- or UDPGlc.When ADPGlc was used as the substrate, the production ofADP is coupled to NADH oxidation via pyruvate kinase(PK) and lactate dehydrogenase (LDH). With UDPGlc assubstrate, UDP was converted to ADP via adenylate kinaseand subsequent coupling to PK and LDH reactions. Usingthis assay, we determined the kinetic parameters of GS andcompared them with those obtained with the classicalradiochemical method. For this purpose, we improved theexpression procedure of A. tumefaciens GS using Escherichiacoli BL21(DE3)-RIL cells. This assay allows thecontinuous monitoring of glycosyltransferase activity usingADPGlc or UDPGlc as sugar-nucleotide donors.