Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, α-D-Glc(1→2)α-D-Glc(1→3)α-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 α-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.
Bibliographical noteFunding Information:
We thank Dr. Rakesh Bhatnagar for technical assistance on confocal laser scanning microscopy. This work was supported by a Strategic Grant (STR 149003 to O.H., N.J.D. and M.M.P.) from the Natural Sciences and Engineering Research Council of Canada. O.H. gratefully acknowledges a Steacie Fellowship from NSERC. N.J.D. gratefully acknowledges a McCalla Professorship from the University of Alberta.
Copyright 2017 Elsevier B.V., All rights reserved.
- Capillary electrophoresis
- In vivo hydrolysis
- Laser induced fluorescence
- Spheroplast single cell analysis