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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.

The activity of ascorbate oxidase (AO) purified from green zucchini squash ( Cucurbita pepo medullosa var. Milano) decreased on freeze-drying. In rehydrated samples, enzyme activity was strictly correlated with the addition of lyoprotectants, the freeze temperature, the enzyme concentration and the storage time. The addition of lyoprotectants such as sucrose, mannose, glucose or polyvinylpyrrolidone (PVP) improved enzyme stabilization. A notable loss of enzymic activity was observed when freezing was performed by immersion in liquid nitrogen in comparison to samples frozen at −20 or −75°C. Moreover, sugar-based lyoprotectants provided better stabilization when the freeze temperature was −75°C. PVP species were most effective in stabilizing AO when samples were frozen in liquid nitrogen. The decrease of enzyme activity on freeze-drying was inversely related to the enzyme concentration. A decrease in enzyme activity was observed in all samples after rehydration at longer storage times, the decrease being less evident in samples containing the lyoprotectants mentioned earlier. Freeze-thawing experiments showed a similar trend considering the presence of lyoprotectant and the enzyme concentration. Samples without additives showed the highest residual activities after the first freeze-thaw cycle but samples were much more prone to a gradual decrease of residual activity with subsequent freeze-thawing cycles.

Purified ascorbate oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native ascorbate oxidase antibodies scarcely recognized deglycosylated protein.