Published: Vol 6, Iss 2, Jan 20, 2016 DOI: 10.21769/BioProtoc.1711 Views: 7366
Reviewed by: Renate WeizbauerHarrie van ErpAnonymous reviewer(s)
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Abstract
Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins regulating a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. The initial Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-galactose (Figure 1). Here we describe a LC/MS-based method for the detection of HPGT activity in vitro.
Figure 1. Reaction scheme for Hyp galactosylation by HPGT. HPGT catalyzes the addition of a D-galactopyranose from an UDP-α-D-Gal to the hydroxylgroup of Hyp residues.
Materials and Reagents
Equipment
Procedure
HPGT assay components | Amount per reaction |
1 M MOPS-KOH (pH 7.0) | 2 μl |
2 mM UDP-α-D-galactose | 5 μl |
10 mM MnCl2 | 2 μl |
10% TX-100 | 2 μl |
2 mM substrate peptide | 1 μl |
Arabidopsis T-87 microsomal membranes | 30 μg protein equivalent |
WaterTotal | 20 μl |
Recipes
Acknowledgments
This is the detailed protocol for the detection of HPGT activity described by Ogawa-Ohnishi and Matsubayashi (2015). This research was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25221105).
References
Article Information
Copyright
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Ogawa-Ohnishi, M. and Matsubayashi, Y. (2016). LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity. Bio-protocol 6(2): e1711. DOI: 10.21769/BioProtoc.1711.
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Carbohydrate > Glycoprotein
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