The glucosylation status and the role of carbohydrate moieties in the heterogeneity of cucumber anionic virus-inducible peroxidase

Three forms of anionic peroxidase (PRX) from hypersensitively reacting cucu mber cotyledons were purified to homogeneity and different methods were use d to analyze the nature of their carbohydrate chains. Immunoblot analysis w ith beta F1 antiserum showed that all three forms are highly glycosylated a nd contain asparagine N-linked glycans commonly found in other plant glycop roteins. Mobility shift analysis showed that chemical deglycosylation conve rted PRXs 1, 2 and 3 to the same-sized (35 K) products. Enzymatic deglycosy lation with a-mannosidase converted PRX1 and PRX2 to immunoreactive product s migrating in mobility shift polyacrylamide gels at the positions of PRX2 and PRX3, respectively. PRX3 treated with alpha -mannosidase yielded a prod uct with Mr similar to that obtained with the chemical deglycosylation. Cle avage of the PRXs 1,2 and 3 by formic acid at the Asp-Pro site resulted in peptide maps and the putative glycopeptides were recognized using PF 1 anti serum. Only one glycopeptide was observed for each of the forms. Lectin-aff inity blot analysis using biotin-conjugated lectins suggested that virus-in ducible PRX contains complex-type N-glycosyl carbohydrate chain(s). These r esults indicate that heterogeneity of cucumber virus-inducible PRX is not c aused mainly by differences in the terminal a-linked mannose residues.