Nonradioactive glycosyltransferase and sulfotransferase assay to study glycosaminoglycan biosynthesis

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-specifi c glycosyltransferases and sulfated by various sulfotransferases. To further our understanding of GAG chain length regulation and synthesis of specifi c 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 specifi c phosphatases, and the released phosphate is subsequently detected using malachite reagents.