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An approach for fluorometric determination of glycosyltransferase activities

  • Glycoconjugate Journal
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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 2N-acetylglucosaminyltransferase, 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.

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Abbreviations

T:

transferase

Gal:

D-N-galactose

GlcNAc:

D-N-acetylglucosamine

GalNAc:

D-N-acetylgalactosamine

HPLC:

high pressure liquid chromatography

UDP:

uridine diphosphate

TES:

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid

pNp:

para-nitrophenyl

NMR:

nuclear magnetic resonance

DMSO:

dimethyl sulphoxide

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Authors and Affiliations

  1. Dipartimento di Biologia Cellulare e Molecolare, Sezione di Biochimica e Biologia Molecolare, University of Perugia, 06126, Perugia, Italy

    Carlo A. Palmerini, Alessandro Datti, Ingrid E. Vanderelst & Aldo Orlacchio

  2. Dipartimento di Chimica, University of Perugia, 06126, Perugia, Italy

    Lucio Minuti

Authors
  1. Carlo A. Palmerini
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  2. Alessandro Datti
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  3. Ingrid E. Vanderelst
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  4. Lucio Minuti
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  5. Aldo Orlacchio
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Palmerini, C.A., Datti, A., Vanderelst, I.E. et al. An approach for fluorometric determination of glycosyltransferase activities. Glycoconjugate J 13, 631–636 (1996). https://doi.org/10.1007/BF00731451

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  • DOI: https://doi.org/10.1007/BF00731451

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