A fluorescent assay for the determination of UDP-GlcNAc: Gal beta 1,3GalNAc-R (GlcNAc to GalNAc) beta 1,6-N-acetylglucosaminyltransferase activity.
A fluorescent assay for UDP-GlcNAc: Gal beta 1,3Gal-NAc-R beta 1,6-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 Gal beta 1,3GalNAc alpha-pAp as the acceptor. The product, Gal beta 1,3(GlcNAc beta 1,6)-GalNAc alpha-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.
Palmerini
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CA
Dipartimento Biologia Cellulare e Molecolare, University of Perugia, Italy.
Datti
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A
Alunni
S
S
VanderElst
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IE
Orlacchio
A
A
D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism; D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D006224:Cricetinae; D003412:Cricetulus; D003619:Dansyl Compounds / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D005690:Galactose; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000032:analysis* / Q000378:metabolism; D009764:o-Phthalaldehyde / Q000737:chemistry
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014312:Trisaccharides; D014537:Uridine Diphosphate N-Acetylglucosamine; D009764:o-Phthalaldehyde; D017351:N-Acetylglucosaminyltransferases; D000116:Acetylgalactosamine; C029038:dansyl chloride; D000117:Acetylglucosamine; D005690:Galactose