Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent-labeled substrate.
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 exo-polygalacturonase activity in the intercellular spaces of cotton cotyledons.
Zhang
Z
Z
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-3035, USA.
Pierce
M L
ML
Mort
A J
AJ
D018548:Cotyledon / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D006026:Glycoside Hydrolases / Q000378:metabolism*; D003368:Gossypium / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism*; D008190:Lyases / Q000378:metabolism*; D009281:Naphthalenes / Q000737:chemistry*; D010368:Pectins / Q000378:metabolism*; D011096:Polygalacturonase / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D006603:Hexuronic Acids; D009281:Naphthalenes; C007819:galacturonic acid; D010368:Pectins; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D006026:Glycoside Hydrolases; D011096:Polygalacturonase; C020136:exopolygalacturonase; D008190:Lyases; C003181:polygalacturonic acid