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Enzyme assay of xylosyltransferase

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Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

Enzyme assay of xylosyltransferase

Authors
 Toshiyasu Koike
Department of Biochemistry, Kobe Pharmaceutical University

Satomi Nadanaka
Department of Biochemistry, Kobe Pharmaceutical University

Hiroshi Kitagawa *
Department of Biochemistry, Kobe Pharmaceutical University
*To whom correspondence should be addressed.
Keywords

proteoglycan

glycosaminoglycan

xylosyltransferase

glycosyltransferase

Reagents
●

UDP-[14C]Xyl (PerkinElmer)

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Synthetic peptide Q-E-E-E-G-S-G-G-G-Q-K (Invitrogen)*

* Synthetic peptides derived from APP (amyloid b A4 precursor protein) [T-E-N-E-G-S-G-L-T-N-I-K] and APLP2 (amyloid b A4 precursor-like protein 2) [S-E-N-E-G-S-G-M-A-E-Q-K] are also reported to be good substrates for XylT in vitro (5).

Instruments
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Superdex peptide HR 10/30 column (GE Healthcare)
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2300TR liquid scintillation counter (PerkinElmer)
Methods
1.

Enzyme assay of XylT-I using the synthetic bikunin analogous peptide, Q-E-E-E-G-S-G-G-G-Q-K, as an acceptor.

1) 

 The reaction mixture for XylT assays (a total volume of 40 μl) contains 10 μl of enzyme preparation (see Comment *), 25 mM MES buffer (pH 6.5), 25 mM KF, 5 mM MgCl2, 5 mM MnCl2, 2 mM UDP-[14C]Xyl (3.0 x 105 dpm), and the synthetic peptide Q-E-E-E-G-S-G-G-G-Q-K (10 nmol).

 Comment 1
2) 

 The enzyme reaction is performed at 37˚C for 16 h.

 Comment 0
3) 

 The 14C-labeled products are separated by gel filtration chromatography on a Superdex peptide column using 0.25 M ammonium bicarbonate containing 7% 1-propanol as the eluent at a flow rate of 0.4 ml/min.

 Comment 0
4) 

 The collected fractions (0.4 ml each) are analyzed by liquid scintillation counter (Fig. 2).

 Comment 0
Figure & Legends

Figure & Legends

 

Fig. 1  Representative scheme for the initiation of GAG synthesis by XylT.

In mammals, UDP-Xyl is synthesized within the lumen of the ER/Golgi. XylT requires UDP-Xyl as a donor substrate. The GAG synthesis is initiated by transfer of a Xyl residue to specific serine residues in the core proteins by different XylTs (XylT-I and XylT-II). Next, GalT-I and GalT-II successively transfer two Gal residues. GlcAT-I completes the biosynthesis of the linkage region by transferring a GlcA residue. SLC35D1, Nucleotide sugar transporter to transport UDP-GlcA (ER membrane); SLC35B4, Nucleotide sugar transporter to transport UDP-Xyl (Golgi membrane); UXS1, UDP-Xyl synthase; GalT, galactosyltransferase; GlcAT, glucuronyltransferase.

 

Fig. 2  Gel filtration analysis of XylT-I reaction products.

14C-labelled reaction products were isolated by gel filtration chromatography on a Superdex peptide column using 0.25M ammonium bicarbonate containing 7% 1-propanol as the eluent at a flow rate of 0.4 ml/min. The separated fractions (0.4 ml each) were measured for radioactivities. Peaks: 1, Q-E-E-E-G-S([14C]Xyl)-G-G-G-Q-K ; 2, UDP- [14C]Xyl.

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Date of registration：2012-11-15 14:54:04
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