Snow D M, Shaper J H, Shaper N L, Hart G W
School of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21207, USA.
Anal Biochem. 1999 Jun 15;271(1):36-42. doi: 10.1006/abio.1999.4104.
We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.
我们开发了一种非放射性方法来检测UDP-半乳糖：β-D-葡萄糖胺β1,4-半乳糖基转移酶（β4GalT-I）的酶活性。采用毛细管电泳结合激光诱导荧光检测（CE-LIF）对异硫氰酸荧光素（FITC）偶联的含O-连接N-乙酰葡糖胺的底物肽和半乳糖封端的产物肽进行基线分离，同时实现低阿托摩尔范围（10⁻¹⁸）的检测水平。向基于硼酸盐的毛细管电泳缓冲液中添加2 mM六亚甲基二胺可调节电渗流，从而实现糖肽产物与反应物的最佳分离。β4GalT-I的活性依赖于锰和UDP-半乳糖的添加。使用该检测方法，我们发现两种预测定位于不同细胞内区室的β4GalT-I构建体，在使用兔网织红细胞转录-翻译系统进行体外表达时具有酶活性。CE-LIF结合体外转录-翻译对产物检测的高灵敏度适用于轻松测定其他新克隆的糖基转移酶的酶活性。
