NDLI: Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.

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Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Gupta, Vineet Patwardhan, Gauri A. Zhang, Qian-Jin Cabot, Myles C. Jazwinski, S. Michal Liu, Yong-Yu
Description	Country affiliation: United States Author Affiliation: Gupta V ( Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, LA, USA.)
Abstract	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.
File Format	HTM / HTML
ISSN	00222275
e-ISSN	15397262
DOI	10.1194/jlr.D002949
Journal	The Journal of Lipid Research
Issue Number	4
Volume Number	51
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2010-04-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Biochemistry Ceramides Enzyme Assays Glucosyltransferases Metabolism 4-chloro-7-nitrobenzofurazan Analogs & Derivatives Animals Cell Line, Tumor Cell Membrane Permeability Chromatography, Thin Layer Colonic Neoplasms Drug Therapy Pathology Enzyme Inhibitors Administration & Dosage Therapeutic Use Antagonists & Inhibitors Genetics Glycosylation Injections, Intralesional Mice Mice, Nude Morpholines Oligonucleotides Ovarian Neoplasms Random Allocation Reproducibility Of Results Xenograft Model Antitumor Assays Comparative Study Research Support, N.i.h., Extramural Research Support, U.s. Gov't, Non-p.h.s. Validation Studies
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Endocrinology

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