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Fluorescent (left panel) and visible light (right) imaging of NCI/ADR-RE tumor cells incubated with NBD C6-Cer, revealing the accumulation of sphingolipid in the cells.
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Glucosylceramide synthase (GCS) catalyzes the first step of glycosphingolipid synthesis by adding glucose residues to ceramide (Cer) to produce glucosylceramide (GlcCer). As GlcCer is a core component of more than 300 different glycolipids, which have diverse roles in physiology and disease, GCS may be a potential biomarker and drug target. However, current enzyme assays typically involve in vitro reactions using prepared enzyme samples, which may not directly correlate with enzyme activity in a cell. In this study, the researchers introduced an approach to determine direct GCS cellular activity using fluorescent NBD C6-ceramide. They were able to separate C6-glucosylceramide from C6-ceramide in cellular extracts using thin-layer chromatography and then quantified the levels by spectrophotometer. This cell-based method is highly sensitive, being able to quantitate values from as little as 1 mg of tissue (~50,000 cells), and the researchers successfully evaluated GCS enzyme activity in multiple cell and tumor samples. The results suggest that this cell-based fluorescent approach would be a simple, reliable and direct means to evaluate GCS in cells and tissues for applications such as predicting drug resistance for cancer.
Direct Quantitative Determination of Ceramide Glycosylation in Vivo: A New Approach to Evaluate Cellular Enzyme Activity of Glucosylceramide Synthase
Vineet Gupta, Gauri A. Patwardhan, Qian-Jin Zhang, Myles C. Cabot, S. Michal Jazwinski and Yong-Yu Liu
J. Lipid Res., published online Oct. 13, 2009