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Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent‐labeled substrate
Author(s) -
Zhang Zhiquan,
Pierce Margaret L.,
Mort Andrew J.
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170214
Subject(s) - in vivo , oligomer , fluorescence , capillary electrophoresis , cotyledon , pectate lyase , biochemistry , in vitro , enzyme , chemistry , enzyme assay , gel electrophoresis , intracellular , substrate (aquarium) , biophysics , chromatography , biology , pectinase , ecology , physics , botany , microbiology and biotechnology , quantum mechanics , organic chemistry
A sensitive assay is described for the detection of pectate‐depolymerizing enzymes using capillary electrophoresis of a fluorescent end‐labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo , such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate‐depolymerizing activity can be inferred from the products. Both endo‐ and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo‐ and exopolygalacturonase activity in the intercellular spaces of cotton cotyledons.