Measurement of glycogen synthase activity in crude extracts by CE
Measurement of glycogen synthase activity in crude extracts by CE
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DOI：
10.1002/elps.200700040
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发表时间：
2007-08-01
期刊：
影响因子：
2.9
通讯作者：
Ureta, Tito
中科院分区：
文献类型：
--
作者：
Wilson, Christian A. M.;Preller, Ana;Ureta, Tito
糖原合酶催化UDP-葡萄糖掺入糖原。酶的活性通常通过分光光度法或放射测定法来测量。第一种方法不适合，因为在粗提物中使用偶联酶存在困难，而第二种方法是一种耗时的方法，涉及糖原分离和放射性操作。我们使用 CZE 技术作为测量糖原合酶活性的新方法。分离在未涂覆的毛细管（53 cm x 50 μm）中以 22 kV（36 μA）进行。样品注射时间为 30 秒，并在 254 nm 处监测核苷酸。在 20 mM 四硼酸盐缓冲液（pH 9.2）中获得最佳分辨率。作为 UDP 和 UDP-葡萄糖浓度函数的吸光度曲线是线性的。卵母细胞提取物中的酶活性与时间（最多 15 分钟）和酶浓度呈线性关系。 K-m（应用程序）。 UDP-葡萄糖的值为 0.87 mM，该值与使用放射测定报告的值相同。 CZE 可以轻松定量化合物、高灵敏度和过程自动化。需要小样本量，避免辅助酶的干扰和放射性操作，并且分析时间显着缩短。
Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the enzyme is usually measured either by a spectrophotometric method or by a radio-assay. The first one is not suitable because of the difficulties regarding the use of coupled enzymes in crude extracts, while the second is a time-consuming method involving glycogen isolation and manipulation of radioactivity. We have used a CZE technique as a novel approach to measure glycogen synthase activity. The separations were performed at 22 kV (36 mu A) in uncoated capillaries (53 cm x 50 mu m). Sample injection time was 30 s and nucleotides were monitored at 254 nm. Best resolution was achieved in 20 mM tetraborate buffer, pH 9.2. Curves of absorbance as a function of UDP and UDP-glucose concentration were linear. Enzyme activity in oocyte extracts was linear with respect to time (up to 15 min) and enzyme concentration. The K-m (app). for UDP-glucose was 0.87 mM, a value identical to the one reported using the radioassay. CZE enables easy quantitation of compounds, high sensitivity, and automation of the process. Small sample sizes are required, interferences by auxiliary enzymes and manipulation of radioactivity are avoided, and analysis time is significantly diminished.