Nonradioactive Glycosyltransferase and Sulfotransferase Assay to Study Glycosaminoglycan Biosynthesis

• DOI: 10.1007/978-1-4939-1714-3_33
• Publication Date: 2014-10-17T13:39:12Z
• Authors: Cheryl M. Ethen; Miranda Machacek; Brittany Prather; Timothy Tatge; Haixiao Yu; Zhengliang L. Wu
• Keywords: Mice; Radioactivity; Animals; Glycosyltransferases; Humans; Reference Standards; Sulfotransferases; Recombinant Proteins; Enzyme Assays; Glycosaminoglycans; Phosphates; 3. Good health

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.