Ethen Cheryl M, Machacek Miranda, Prather Brittany, Tatge Timothy, Yu Haixiao, Wu Zhengliang L
Department of Enzyme, R&D Systems, 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
Methods Mol Biol. 2015;1229:431-41. doi: 10.1007/978-1-4939-1714-3_33.
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.
糖胺聚糖（GAGs）是具有重复二糖单元的线性多糖。GAGs包括肝素、硫酸乙酰肝素、硫酸软骨素、硫酸皮肤素、硫酸角质素和透明质酸。除透明质酸外，所有GAGs通常都被硫酸化。GAGs由单特异性或双特异性糖基转移酶聚合，并由各种硫酸转移酶硫酸化。为了进一步了解GAG链长度调节和GAG链上特定硫酸化基序的合成，必须了解GAG合成酶的动力学。在此，描述了用于这些糖基转移酶和硫酸转移酶的非放射性比色酶法。在这两种情况下，使用特定的磷酸酶水解离去的核苷酸或核苷，随后使用孔雀石试剂检测释放的磷酸盐。
