title: Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.
abstract: Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.
authors.affiliation: Department of Applied and Basic Biology, University of L'Aquila, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Pitari
authors.forename: G
authors.initials: G
authors.lastname: D'Andrea
authors.forename: M L
authors.initials: ML
authors.lastname: Salucci
authors.forename: A
authors.initials: A
authors.lastname: Rossi
authors.forename: L
authors.initials: L
authors.lastname: Avigliano
mesh_terms: D001204:Ascorbate Oxidase / Q000096:biosynthesis* / Q000276:immunology; D002478:Cells, Cultured; D019662:Cucurbitaceae / Q000166:cytology / Q000201:enzymology*; D006031:Glycosylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D014415:Tunicamycin / Q000494:pharmacology*
chemical_list: D014415:Tunicamycin; D001204:Ascorbate Oxidase