A new strategy for the fluorometric determination of glycosyltransferase activities is reported. The method involves dansyl chloride derivatization of the reduced form (pNH2phenyl) of a hydrophobic, aglycon moiety covalently linked to a number of acceptor substrates (pNO2phenyl). Focusing on the Golgi enzyme core 2 N-acetyl-glucosaminyltransferase, we found that synthesis and fractionation of the dansylated substrate derivative were rapid, easy and inexpensive. Additionally, the corresponding enzyme assay proved reproducible and very sensitive, as 0.4 pmol of reaction product were readily detected. This fluorometric approach appears therefore to be a valid tool for investigating and monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.
An approach for fluorometric determination of glycosyltransferase activities
PALMERINI, Carlo Alberto;DATTI, Alessandro;MINUTI, Lucio;ORLACCHIO, Aldo
1996
Abstract
A new strategy for the fluorometric determination of glycosyltransferase activities is reported. The method involves dansyl chloride derivatization of the reduced form (pNH2phenyl) of a hydrophobic, aglycon moiety covalently linked to a number of acceptor substrates (pNO2phenyl). Focusing on the Golgi enzyme core 2 N-acetyl-glucosaminyltransferase, we found that synthesis and fractionation of the dansylated substrate derivative were rapid, easy and inexpensive. Additionally, the corresponding enzyme assay proved reproducible and very sensitive, as 0.4 pmol of reaction product were readily detected. This fluorometric approach appears therefore to be a valid tool for investigating and monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
