Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells

Abstract

Seedlings and suspension-cultured cells of carrot (Daucus carota) contain a cell wall associated as well as a soluble form of beta-fructosidase (beta F). These two forms have different pH optima: 4.6 for cell wall beta F and 5.6 for soluble beta F. Soluble beta F is relatively more abundant in the seedlings and cell wall beta F is relatively much more abundant in the cultured cells. Protoplasts of cultured cells have only the soluble form (pH optimum 5.6) indicating that the cell wall associated form is indeed extracellular in situ. Cell wall beta F was purified to homogeneity and has an Mr = 63,000. Antibodies raised against the deglycosylated enzyme cross-reacted with two soluble enzyme forms: in cultured cells, the soluble enzyme has an Mr = 58,000 and, in seedlings, there are two forms of Mr = 58,000 and 52,000. Treatment of purified cell wall beta F with endoglycosidase H and trifluoromethanesulfonic acid (complete deglycosylation) indicated that the enzyme probably has one high mannose and two complex glycans. This was confirmed by HPLC analysis of [3H]GlcNAc- and [3H]fucose-labeled glycopeptides obtained after trypsin digestion of radioactively-labeled beta F. The amino acid composition shows that cell wall beta F has 18.6% glycine.