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 μg 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 μg ml−1 tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 μg 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 μg 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.
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Pitari, G., D'Andrea, G., Salucci, M.L. et al. Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells. Glycoconj J 15, 777–782 (1998). https://doi.org/10.1023/A:1006943412709
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DOI: https://doi.org/10.1023/A:1006943412709