L Avigliano

Immunohistochemical localization of ascorbate oxidase in cucurbita pepo medullosa

Abstract Antibodies raised against homogeneous ascorbate oxidase (AAO) were used for the immunohistochemical localization of the enzyme in Cucurbita pepo medullosa. Ascorbate oxidase is present in all the specimens examined (vegetative and reproductive organs). At the cellular level the enzyme is associated with the cell wall and cytoplasm. The ubiquitous distribution of ascorbate oxidase suggest a role of general relevance for plant cells.

Activity and expression of diamine oxidase in lentil seedling under different growth conditions

The activity and expression of diamine oxidase during the germination of lentil seeds and in the course of anoxic and thermal stress have been studied. Diamine oxidase activity, as well as the seedlings growth rate, was found to be markedly higher in dark-grown lentil seedlings than in the light-grown ones. The same was true for the respective protein and mRNA amounts. The specific activity of diamine oxidase was decreased by anoxic stress and not affected by thermal stress. The possible physiological meaning of these findings is discussed.

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium.

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.

Efficiency of nitrilotriacetate in the removal of type 2 copper from laccase and ascorbate oxidase

Abstract The powerful chelating agent nitrilotriacetate was found to be quite efficient in the removal of type 2 Cu, under reducing conditions, from the blue oxidases ascorbate oxidase and Rhus vernicifera laccase. While the effect of this substance on ascorbate oxidase was comparable to that of other reagents, as for instance EDTA, the effect on laccase was substantially larger than that of any other previously tested chelator, in particular EDTA which is by itself ineffective. This result confirms that the size of the chelator strongly affects its reactivity with laccase type 2 Cu, suggesting that the latter ion is more deeply buried in the protein matrix than the copper of ascorbate oxidase.

Characterization and cross-reactivity of antibodies against ascorbate oxidase from green zucchini marrows (Cucurbita pepo medullosa)

Abstract Antibodies have been elicited in rabbits against homogeneous ascorbate oxidase from Cucurbita pepo medullosa . These antibodies were found to inhibit the enzyme activity in a non-competitive way. They reacted faster with copper-free ascorbate oxidase but more slowly with a deglycosylated enzyme sample. Homogenates from some other plants show cross-reaction with these antibodies. The same homogenates had also an ascorbate oxidase activity roughly comparable with the extent of immunological cross-reactivity.

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 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.