Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels.
The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M(r) polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.
Farkas
Vladimír
V
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84538 Bratislava, Slovakia. chemvfar@savba.sk
Ait-Mohand
Fairouz
F
Stratilová
Eva
E
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D016695:Glycosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing / Q000379:methods*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D015203:Reproducibility of Results; D019685:Rosales / Q000201:enzymology
D007527:Isoenzymes; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase; D006867:Hydrolases