An approach for fluorometric determination of glycosyltransferase activities.
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 the monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.
Palmerini
C A
CA
Dipartimento di Biologia Cellulare e Molecolare, University of Perugia, Italy.
Datti
A
A
Vanderelst
I E
IE
Minuti
L
L
Orlacchio
A
A
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D009844:Oligosaccharides; D016695:Glycosyltransferases