Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase
Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase
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DOI：
10.1194/jlr.d002949
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发表时间：
2010-04-01
影响因子：
6.5
通讯作者：
Liu, Yong-Yu
中科院分区：
文献类型：
--
作者：
Gupta, Vineet;Patwardhan, Gauri A.;Liu, Yong-Yu
葡萄糖神经酰胺合成酶(GCS或GlcT-1)将神经酰胺转化为葡萄糖神经酰胺，是神经鞘糖脂合成的关键酶。由于其在生理和疾病中的不同作用，GCS可能成为疾病的标志物和药物靶点。目前对包括GCS在内的酶的分析是基于使用酶制剂在试管中进行的反应。实验室条件下的酶活性测量不能直接评估GCS在细胞中的作用。在这里，我们介绍了一种新的方法来确定GCS细胞活性的荧光NBD C6神经酰胺在体内。细胞GCS将UDP-葡萄糖转移到NBD C6-神经酰胺，并产生NBD C6-葡萄糖神经酰胺。然后用薄层层析法分离C6-神经酰胺和C6-神经酰胺，然后用分光光度计定量。这种基于细胞的方法能够定量测定大约50,000个细胞或1.0 mg组织产生的pmoL范围内的葡萄糖神经酰胺。该方法已成功地用于评估细胞和受基因操作和化学抑制的肿瘤中GCS酶的程度。这些数据表明，这种基于细胞的荧光方法是评估神经酰胺糖基化的直接、重复性和简单的方法。它适用于验证GCS在耐药癌症和其他疾病中的活性。首页--期刊主要分类--期刊细介绍--期刊题录与文摘--文摘内容刘先生。体内神经酰胺糖基化的直接定量测定：一种评价葡萄糖神经酰胺合成酶细胞酶活性的新方法。J.Lipid Res.2010年。51：866-874。
Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.-Gupta, V., G. A. Patwardhan, Q-J. Zhang, M. C. Cabot, S. M. Jazwinski, and Y-Y. Liu. Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase. J. Lipid Res. 2010. 51: 866-874.