Chunfang Hu

Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Peroxidase isozymes are distributed throughout the plant kingdom. In order to understand the functions of the isoperoxidases, the nature and degree of the heterogeneity between them need to be clarified. The cationic (C.PRX) and the anionic peanut peroxidases (A.PRX) are used for such a study.;The physico-chemical properties of the two peroxidases are reported. They are similar in MW, carbohydrate content and pH optimum for catalysis, but differ in Ca{dollar}\sp{lcub}2+{rcub}{dollar} contents and isoelectric point. The basis for the difference in pI is found in the different relative content of the acidic and basic amino acids, with more acidic residues present in the anionic and more basic ones in the cationic isozyme.;Polyclonal antibodies generated to each isozyme and monoclonal antibodies to the C.PRX were used to probe the antigenic structures of the two peroxidases by double-immunodiffusion, ELISA, immunoinhibition of enzyme activity and Western blotting. The polyclonal antisera reacted not only with the homologous but also with the non-homologous isozyme, suggesting similarities in antigenic structure exist between the two peroxidases. The cross-reaction is partially attributed to the epitopes present in the carbohydrate moieties and near the active sites of the isozymes. On the other hand, the monoclonal antibodies recognized more specifically the C.PRX.;Radiolabeling of peanut cell cultures coupled with immunoprecipitation reveals that four times as much C.PRX is released into the medium as the A.PRX, although no substantial difference was observed in the rate of their synthesis. This release is not prevented by treatment of the cultures with tunicamycin, an inhibitor of N-linked glycosylation. Subcellular localization by immunogold-labeling with their antibodies demonstrates that both peroxidases are associated with Golgi complexes and amyloplasts. The C.PRX is also found on the cell walls while the A.PRX on the plasmalemma.;Evidence obtained from deglycosylation with glycopeptidase F, oxidation of carbohydrates with periodate and inhibition of glycosylation with tunicamycin indicates that the carbohydrate side chains distributed on the surface of the peroxidases are essential for expression of the enzyme activity and for maintenance of the stability of the molecules.



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