Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tis sue extracts obtained from callus cell cultures induced by green zucchini s arcocarp and grown in the presence of tunicamycin, a powerful N-glycosylati on inhibitor. Tunicamycin at 2 or 4 mu g 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 observ ed at 10 and 15 days of culture time, with a decrease of 30% and 43% respec tively when cells were grown in the presence of 2 mu g ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 mu g 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 mu g ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the sa me growing times. Lectin-blot, based on the use of concanavalin A, indicate d a marked decrease of glycosylated proteins in tunicamycin-treated culture s. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarce ly recognized the enzyme expressed in tunicamycin-treated cells; on the con trary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.