Cell Wall and Cytoplasmic Isozymes of Radish beta-Fructosidase Have Different N-Linked Oligosaccharides

When 36-hour-old dark grown radish seedlings are transferred to far-red light, there is a decrease in cytoplasmic β-fructosidase (βF) and an increase in cell wall βF compared to the dark controls. Cytoplasmic and cell wall-bound β-fructosidase are both glycoproteins and exhibit high antigenic similarities, but differ according to charge heterogeneity and carbohydrate microheterogeneity. Growth of radish seedlings in the presence of tunicamycin results in a partial inhibition of βF glycosylation but nonglycosylated βF still accumulates in the cell wall under far-red light. Thus, glycosylation is not necessary for intracellular transport, for correct targetting, or for wall association of an active βF. The nonglycosylated cytoplasmic and cell wall βF forms have the same relative molecular mass but glycosylated forms have different oligosaccharide side-chains, with respect to size and susceptibility to α-mannosidase and endoglycosidase D digestion. The oligosaccharides of both forms are partly removed by endoglycosidase H when βF is denatured. Isoelectric focusing analysis of βF shows that the cell wall-associated isozymes are more basic than the cytoplasmic isozymes, and that the charge heterogeneity also exists within a single plant. A time course of changes in βF zymograms shows a far red light stimulation of the appearance of the basic forms of the enzyme. However, the more basic cell wall specific βF forms are not present when N-glycosylation is prevented with tunicamycin. These results indicate that cytoplasmic and cell wall βF probably have common precursor polypeptides and basic cell wall forms arise via processing events which are tunicamycin sensitive.