A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

Biological Procedures Online

Peng Wang¹  Xiaofeng Ma²  Jing Li²  Feifei Ren²  Lianwen Zhang² 

[1]Departments of Biochemistry and Chemistry, the Ohio State University, Columbus, OH, 43210, USA

[2]College of Pharmacy, Nankai University, Tianjin, 300071, People's Republic of China

关键词: kinetic study;    MS-glycosylation assay;    coupled enzyme method;    O-linked GlcNAc transferase (OGT);   

Others  :  797147
DOI  :  10.1007/s12575-009-9016-x

 received in 2009-06-06, accepted in 2009-08-13,  发布年份 2009

【 摘 要 】

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.

【 授权许可】

   
2009 Zhang et al.

【 图 表 】

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