Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity.
Purified ascorbate oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native ascorbate oxidase antibodies scarcely recognized deglycosylated protein.
D'Andrea
G
G
Department of Biomedical Sciences, University of L'Aquila, Collemaggio, Italy.
Salucci
M L
ML
Pitari
G
G
Avigliano
L
L
D001204:Ascorbate Oxidase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006031:Glycosylation; D007700:Kinetics; D011134:Polysaccharides / Q000302:isolation & purification / Q000378:metabolism*; D014675:Vegetables / Q000201:enzymology
D011134:Polysaccharides; D001204:Ascorbate Oxidase