R&D Systems Inc., Minneapolis, MN 55413, USA.
Glycobiology. 2011 Jun;21(6):727-33. doi: 10.1093/glycob/cwq187. Epub 2010 Nov 15.
A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multiwell plates and quantitated by a plate reader, thus making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. As examples, we first assayed Clostridium difficile toxin B, a protein O-glucosyltransferase that specifically monoglucosylates and inactivates Rho family small GTPases; we then showed that human KTELC1, a homolog of Rumi from Drosophila, was able to hydrolyze UDP-Glc; and finally, we measured the kinetic parameters of human sialyltransferase ST6GAL1.
这里描述了一种非放射性糖基转移酶测定法。该方法利用特定的磷酸酶，这些磷酸酶可以被添加到糖基转移酶反应中，从而从糖基转移酶反应的离去基团中定量释放无机磷酸。然后使用比色法基于孔雀石绿的试剂检测释放的磷酸基团。因为释放的磷酸基团的量与糖基转移酶反应中转移的糖分子直接成正比，所以该方法可用于获得糖基转移酶的准确动力学参数。该测定法可以在多孔板中进行，并通过板读数器进行定量，因此适用于高通量筛选。它已成功应用于我们可获得的所有糖基转移酶，包括葡萄糖基转移酶、N-乙酰氨基葡萄糖基转移酶、N-乙酰半乳糖基转移酶、半乳糖基转移酶、岩藻糖基转移酶和唾液酸转移酶。作为示例，我们首先测定了艰难梭菌毒素 B，一种特异性单葡糖基化并使 Rho 家族小分子 GTP 酶失活的蛋白 O-糖基转移酶；然后表明，人类 KTELC1，一种来自果蝇的 Rumi 同源物，能够水解 UDP-Glc；最后，我们测量了人唾液酸转移酶 ST6GAL1 的动力学参数。
