> I have noticed that one can use commercially available AMC-labelled oligosaccharides as substrates
> for glycosidase assays.
> My question is whether there is any experience around with using similarly labeelled
> oligosaccharides as substrates for glycosyltransferases. If yes then how far the label should
> be from the reaction centre and what might be the optimal method for following the reaction, e.g.
> TLC or HPLC.I am mostly thinking about assaying the activity of alpha- galactosyl transferase.
>> Thanks,
> Armin Sepp, PhD
>Armin.Sepp at bbsrc.ac.uk
In our lab pyridylamino-oligosaccharides have been used as substrates
for GlcNAc transferases - e.g. Man5GlcNAc2-PA and Man3GlcNAc2-PA. See
Altmann et al (1993) Glycobiology 3, 619-625. The oligosaccharides
were subsequently separated on HPLC (Hypersil ODS) and fluorescence
was monitored (320/400 nm). If you're thinking of alpha1,3-GalT use
of an asialo-biantennary N-link with PA label should be possible,
since GlcNAc-transferases act before alpha1,3GalT. Consider also that
terminal glycosyltransferases appear tolerant of a range of
octyloligosaccharides that lack the chitobiose portion of N-linked
oligosaccharides.
Iain
---------------------------------------------------------------
Iain Wilson Institut fuer Chemie
Tel: 43-1-47654-6065 Universitaet fuer Bodenkultur
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E-mail: wilson at edv1.boku.ac.at A-1180, WIEN, Austria
