Reaktion #2339697

Reaktionsgleichung

UDP-GlcNAc

HEPES

α-GalNAc

→

N-acetylglucosamine

Lösungsmittel

acetonitrile

Reaktionsbedingungen

Aufarbeitung

1. 1
   workup.ADDITIONto which 5 μl of β3Gn-T6 was added
2. 2
   SonstigeAfter completion of the reaction, analysis
3. 3
   SonstigeH2O=10:90, and the detection was carried out in terms of the absorption at 210 nm
4. 4
   WaschenAs a result, as for the standard samples, GalNAcα-pNp was eluted at 23.5 minutes
5. 5
   WaschenGlcNAcβ1-3GalNAcα-pNp was eluted at 18.6 minutes
6. 6
   WaschenGlcNAcβ1-6GalNAcα-pNp was eluted at 20 minutes
7. 7
   workup.WAITAs for the β3Gn-T6 solution, the peak at 23.5 minutes (GalNAcα-pNp)
8. 8
   workup.WAITthe peak at 18.6 minutes (GlcNAcβ1-3GalNAcα-pNp) were observed

Vorschrift

The reaction solution contained 50 mM HEPES buffer, 10 mM MnCl2, 0.1% Triton-CF54, 1 mM pNp-α-GalNAc and 1.5 mM UDP-GlcNAc, to which 5 μl of β3Gn-T6 was added, so as to have a total volume of 20 μl. The reaction was carried out at 37° C. for 16 hours. After completion of the reaction, analysis was carried out using HPLC. The column used was Mightysil RP-18, 250×4 mm, the solvent was acetonitrile: H2O=10:90, and the detection was carried out in terms of the absorption at 210 nm. As standard samples, GlcNAcβ1-3GalNAcα-pNp (core 3 structure) and GlcNAcβ1-6GalNAcα-pNp (core 6 structure) were used (SIGMA). As a result, as for the standard samples, GalNAcα-pNp was eluted at 23.5 minutes, GlcNAcβ1-3GalNAcα-pNp was eluted at 18.6 minutes and GlcNAcβ1-6GalNAcα-pNp was eluted at 20 minutes. As for the β3Gn-T6 solution, the peak at 23.5 minutes (GalNAcα-pNp) and the peak at 18.6 minutes (GlcNAcβ1-3GalNAcα-pNp) were observed. Thus, β3Gn-T6 is a novel enzyme having an activity to transfer N-acetylglucosamine to a non-reducing terminal of N-acetylgalactosaminyl group through the β1,3-linkage.

Quelle