Article

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

Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, LA, USA.
The Journal of Lipid Research (impact factor: 5.56). 10/2009; 51(4):866-74. DOI:10.1194/jlr.D002949 pp.866-74
Source: PubMed

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.

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Keywords

C6-glucosylceramide
 
cell-based fluorescent method
 
cell-based method
 
Cellular GCS transfers UDP-glucose
 
chemical inhibition
 
disease marker
 
drug-resistant cancers
 
fluorescent NBD C6-ceramide
 
GCS cellular activity
 
GCS enzyme
 
gene manipulation
 
glucosylceramide
 
Glucosylceramide synthase
 
key enzyme
 
NBD C6-ceramide
 
NBD C6-glucosylceramide
 
pmol range
 
quantitate glucosylceramide
 
thin-layer chromatography
 
validate GCS activity