Search

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.

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Nothern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.

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.