ABSTRACT Nondenaturing gel electrophoresis allows the separation and analysis of biological enzymes while preserving their activity and substrate‐binding capabilities. The activity of carboxylesterases separated by nondenaturing two‐dimensional electrophoresis was detected using the fluorescent substrate, 4‐methylumbelliferyl acetate, facilitating subsequent reseparation and further analysis by nondenaturing electrophoresis. In addition, by performing nondenaturing three‐dimensional electrophoresis with concanavalin A, these carboxylesterases were identified as carbohydrate‐binding enzymes capable of interacting with concanavalin A. Enzyme activity of the concanavalin A‐bound fraction was diminished upon treatment with peptide N‐glycosidase F, which releases N‐glycans. Following capture with concanavalin A and treatment with methyl‐α‐D‐mannopyranoside, carboxylesterase activity was further analyzed using nondenaturing four‐dimensional electrophoresis. These approaches are applicable to enzymes that can be separated by nondenaturing two‐dimensional electrophoresis and whose activity is detectable using colorimetric and fluorescent dyes, because concanavalin A within the gel exhibits autofluorescence. Collectively, these finding demonstrate that combining multidimensional nondenaturing electrophoresis with concanavalin A provides a powerful approach to investigate the glycosylation status of enzymes.
Shimazaki et al. (Wed,) studied this question.