Purification of beta-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis.
The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I.
Wójczyk
B
B
Department of Animal Physiology, Jagiellonian University, Kraków, Poland.
Hoja
D
D
Lityńska
A
A
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry*; D002846:Chromatography, Affinity; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005966:Glucuronidase / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar
D002241:Carbohydrates; D003208:Concanavalin A; D005966:Glucuronidase