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Glycobiology, Vol 9, 219-225, Copyright © 1999 by Oxford University Press

ORIGINAL ARTICLES

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

XC Le, W Tan, CH Scaman, A Szpacenko, E Arriaga, Y Zhang, NJ Dovichi, O Hindsgaul and MM Palcic
Department of Public Health Sciences, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-- >2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.
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