影响N—乙酰氨基葡萄糖转移酶Ⅴ活性测定因素的研究

THE EFFECTS OF SOME FACTORS ON THE ACTIVITY OF N-ACETYLGLUCOSAMINYLTRANSFERASⅤ

目的探讨诸多因素对Ｎ—乙酰氨基葡萄糖转移酶（ＧｎＴ）Ⅴ活性测定的影响。方法采用反相ＨＰＬＣ荧光法测定ＧｎＴⅤ活性时的一些影响因素，包括反应时间、酶蛋白量、ｐＨ和去垢剂等。结果反应时间在８ｈ以内，ＧｎＴⅤ产物的生成量与时间呈正比；酶蛋白量在３００μｇ以下，产物的生成量与酶量呈正比；ＧｎＴⅤ的最适ｐＨ为６．２５；ＴｒｉｔｏｎＸ－１００对ＧｎＴⅤ有明显增溶激活作用，其最适浓度为１．０％；ＧｌｃＮＡｃ对ＧｎＴⅤ活性影响不明显。结论ＧｎＴⅤ活性测定受诸多因素的影响。测定时应注意保持测定条件的稳定和统一。

Objective To study the effects of some factors on the activity of N-acetylglucosaminyltransferaseⅤ.Methods By using reverse-phase HPLC and fluorescence labeled substrate,the effect of some factors on GnT V activity was investigated,including enzyme protein concentration,incubation time,pH and detergent.Results The GnT V activity was proportioned to the enzyme protein concentration at range of 0-300 μg and the products produced by GnT V were linear with incubation time up to 8 hr..GnT V displayed a pH optimum at 6.25.The optimum concentration of Triton X-100 for activation of GnT V was 1.0%.The effect of GlcNAc on GnT V activity was of no significance.Conclusion The activity of N-acetylglucosaminyltransferase V was affected by some factors,so the assay condition should be kept consistent