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MS analysis of chondroitin polymerization: effects of Mn2+ ions on the stability of UDP-sugars and chondroitin synthesis.
- Source :
-
Analytical biochemistry [Anal Biochem] 2007 Jun 01; Vol. 365 (1), pp. 62-73. Date of Electronic Publication: 2007 Feb 24. - Publication Year :
- 2007
-
Abstract
- Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn(2+) ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn(2+) ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.
- Subjects :
- Carbohydrate Sequence
Catalysis
Cations metabolism
Chondroitinases and Chondroitin Lyases metabolism
Hexosyltransferases metabolism
Hydrolysis
Kinetics
Manganese metabolism
Models, Chemical
Molecular Weight
Substrate Specificity
Uridine Diphosphate Glucuronic Acid metabolism
Uridine Diphosphate N-Acetylgalactosamine metabolism
Uridine Diphosphate N-Acetylglucosamine metabolism
Cations chemistry
Chondroitin biosynthesis
Hexosyltransferases analysis
Manganese chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Uridine Diphosphate Sugars chemistry
Uridine Diphosphate Sugars metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0003-2697
- Volume :
- 365
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17395146
- Full Text :
- https://doi.org/10.1016/j.ab.2007.02.023