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

X. Chris Le; Woei Tan; Christine H. Scaman; Adam Szpacenko; Edgar Arriaga; Yanni Zhang; Norman J. Dovichi; Ole Hindsgaul; Monica M. Palcic

doi:10.1093/glycob/9.3.219

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

X. Chris Le, Woei Tan, Christine H. Scaman, Adam Szpacenko, Edgar Arriaga, Yanni Zhang, Norman J. Dovichi, Ole Hindsgaul, Monica M. Palcic

Chemistry (Twin Cities)

Research output: Contribution to journal › Article › peer-review

35 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	219-225
Number of pages	7
Journal	Glycobiology
Volume	9
Issue number	3
DOIs	https://doi.org/10.1093/glycob/9.3.219
State	Published - Mar 1999

Bibliographical note

Funding 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:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

Capillary electrophoresis
In vivo hydrolysis
Laser induced fluorescence
Spheroplast single cell analysis
α-glucosidase

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10.1093/glycob/9.3.219

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title = "Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast",

abstract = "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.",

keywords = "Capillary electrophoresis, In vivo hydrolysis, Laser induced fluorescence, Spheroplast single cell analysis, α-glucosidase",

author = "Le, {X. Chris} and Woei Tan and Scaman, {Christine H.} and Adam Szpacenko and Edgar Arriaga and Yanni Zhang and Dovichi, {Norman J.} and Ole Hindsgaul and Palcic, {Monica M.}",

note = "Funding 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: Copyright 2017 Elsevier B.V., All rights reserved.",

year = "1999",

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doi = "10.1093/glycob/9.3.219",

language = "English (US)",

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AU - Szpacenko, Adam

AU - Arriaga, Edgar

AU - Zhang, Yanni

AU - Dovichi, Norman J.

AU - Hindsgaul, Ole

AU - Palcic, Monica M.

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PY - 1999/3

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N2 - 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.

AB - 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.

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KW - Laser induced fluorescence

KW - Spheroplast single cell analysis

KW - α-glucosidase

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

Abstract

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