A fluorescent assay for UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-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 Galbeta1,3GalNAcalpha-pAp as the acceptor. The product, Galbeta1,3(GlcNAcbeta1,6)-GalNAcalpha-pAp, was quantitatively derivatized with dansyl chloride at the NH2 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 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.
A Fluorescent Assay for the Determination of UDP-GlcNAc: Galβ1,3GalNAc-R (GlcNAc to GalNAc) β-1,6 N-Acetylglucosaminyltransferase Activity
PALMERINI, Carlo Alberto;DATTI, Alessandro;ORLACCHIO, Aldo
1995
Abstract
A fluorescent assay for UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-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 Galbeta1,3GalNAcalpha-pAp as the acceptor. The product, Galbeta1,3(GlcNAcbeta1,6)-GalNAcalpha-pAp, was quantitatively derivatized with dansyl chloride at the NH2 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 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
