期刊
BIOLOGICAL PROCEDURES ONLINE
卷 11, 期 1, 页码 170-183出版社
BIOMED CENTRAL LTD
DOI: 10.1007/s12575-009-9016-x
关键词
O-linked GlcNAc transferase (OGT); coupled enzyme method; MS-glycosylation assay; kinetic study
In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E-m = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.
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