Naoi M, Kondoh M, Mutoh T, Takahashi T, Kojima T, Hirooka T, Nagatsu T
Department of Biochemistry, Nagoya University School of Medicine, Japan.
J Chromatogr. 1988 Apr 8;426(1):75-82. doi: 10.1016/s0378-4347(00)81928-3.
A simple and sensitive assay for GM1 ganglioside (GM1) beta-galactosidase activity was devised by direct measurement of released D-galactose using high-performance liquid chromatography (HPLC). GM1 beta-galactosidase activity in crude samples such as brain homogenates could be measured by this method. After incubation of brain homogenate for 1 h with GM1 at 37 degrees C and pH 4.4 in the presence of sodium taurodeoxycholate, the reaction was terminated by heating at 100 degrees C for 2 min and the supernatant from the centrifuged sample was analysed directly by HPLC. D-Galactose isolated by HPLC was converted into a fluorescent compound by a post-column reaction with arginine at 150 degrees C and the fluorescence intensity at 430 nm was measured with excitation at 320 nm. By this method 10 pmol of D-galactose could be measured and the fluorescence intensity was linear up to 1 mmol of D-galactose. Using this method, the optimal conditions for the activity of this enzyme were re-examined. As an application, the enzyme activity in the brain of a patient with GM1 gangliosidosis was examined. This method can be applied to any natural substrates, glycolipids or glycoproteins, the terminal galactose of which is hydrolysed by this enzyme.
通过使用高效液相色谱法（HPLC）直接测量释放的D-半乳糖，设计了一种简单灵敏的GM1神经节苷脂（GM1）β-半乳糖苷酶活性测定方法。用这种方法可以测量粗样品如脑匀浆中的GM1β-半乳糖苷酶活性。在牛磺脱氧胆酸钠存在下，将脑匀浆与GM1在37℃和pH 4.4下孵育1小时后，通过在100℃加热2分钟终止反应，离心样品的上清液直接用HPLC分析。通过HPLC分离的D-半乳糖在150℃与精氨酸进行柱后反应转化为荧光化合物，并在320nm激发下测量430nm处的荧光强度。用这种方法可以测量10pmol的D-半乳糖，荧光强度在高达1mmol的D-半乳糖范围内呈线性。使用这种方法，重新检查了该酶活性的最佳条件。作为应用，检测了一名GM1神经节苷脂沉积症患者大脑中的酶活性。该方法可应用于任何天然底物、糖脂或糖蛋白，其末端半乳糖可被该酶水解。
