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Nonradioactive Glycosyltransferase and Sulfotransferase Assay to Study Glycosaminoglycan Biosynthesis

Part of the Methods in Molecular Biology book series (MIMB,volume 1229)

Abstract

Glycosaminoglycans (GAGs) are linear polysaccharides with repeating disaccharide units. GAGs include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. All GAGs, except for hyaluronan, are usually sulfated. GAGs are polymerized by mono- or dual-specific glycosyltransferases and sulfated by various sulfotransferases. To further our understanding of GAG chain length regulation and synthesis of specific sulfation motifs on GAG chains, it is imperative to understand the kinetics of GAG synthetic enzymes. Here, nonradioactive colorimetric enzymatic assays are described for these glycosyltransferases and sulfotransferases. In both cases, the leaving nucleotides or nucleosides are hydrolyzed using specific phosphatases, and the released phosphate is subsequently detected using malachite reagents.

Key words

  • Glycosyltransferase assay
  • Sulfotransferase assay
  • Glycosaminoglycans
  • Enzyme kinetics
  • Heparin
  • Heparan sulfate
  • Chondroitin sulfate
  • Keratan sulfate
  • Hyaluronan

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Acknowledgement

This work was supported by R&D Systems, and we would like to thank all our colleagues who made contributions through product development.

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Correspondence to Zhengliang L. Wu .

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Ethen, C.M., Machacek, M., Prather, B., Tatge, T., Yu, H., Wu, Z.L. (2015). Nonradioactive Glycosyltransferase and Sulfotransferase Assay to Study Glycosaminoglycan Biosynthesis. In: Balagurunathan, K., Nakato, H., Desai, U. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 1229. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1714-3_33

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  • DOI: https://doi.org/10.1007/978-1-4939-1714-3_33

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  • Publisher Name: Humana Press, New York, NY

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  • Online ISBN: 978-1-4939-1714-3

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