A Fluorescent Assay for the Determination of UDP-GlcNAc: Galβ1,3GalNAc-R (GlcNAc to GalNAc) β-1,6 N-Acetylglucosaminyltransferase Activity

✍ Scribed by C.A. Palmerini; A. Datti; S. Alunni; I.E. Vanderelst; A. Orlacchio

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 429 KB
• Volume: 225
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1160

✦ Synopsis

A fluorescent assay for UDP-GlcNAc: Galp1,3GalNAc-R (\boldsymbol{\beta 1 , 6 - \boldsymbol { N } \text { -acetylglucosaminyltransferase (core } 2}) GlcNAc-T) activity has been developed involving dansylation of the enzyme reaction product. Core 2 GlcNAc-T detection was performed using unlabeled UDP-GlcNAc as the donor and Gal (\beta 1,3 \mathrm{GalNAc} \alpha-) pAp as the acceptor. The product, (\mathrm{Gal} \beta 1,3(\operatorname{GlcNAc} \beta 1,6)) GalNAc (\alpha)-pAp, was quantitatively derivatized with dansyl chloride at the (\mathbf{N H}_{2}) moiety of the pAp group and the resultant fluorescent trisaccharide was separated on a Spherisorb ODS2 HPLC column. This method, rapid and economical, was found to be sensitive enough for the detection of (1 \mathrm{pmol}) of reaction product and therefore represents a reliable alternative to assays which use radiolabeled substrates. Additionally, the approach described here can be adapted for the assay of other glycosyltransferases, where the acceptor substrate has a pAp group as a hydrophobic aglycon linker. c 1995 Academic Press, Inc.