ISSN:
1573-4986
Keywords:
ascorbate oxidase
;
tunicamycin
;
cell culture
;
AO
;
Con A, concanavalin A
;
G-6-PDH, glucose-6-phosphate dehydrogenase
;
PAGE, polyacrylamide gel electrophoresis
;
SDS, sodium dodecyl sulfate
;
TM
;
TBS, Tris-buffered saline: 500 m M NaCl, 20 m M Tris-HCl
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1006943412709
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