Demonstration of Activity ofα-Galactosidase Secreted byCucumis sativus L. Cells

Abstract

A simple, rapid and reproducible procedure for the identification of extracellular cucumber (Cucumis sativus L.) α-galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2-day-old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α-galactosidase, 6-bromo-2-naphthyl--α--D-galactopyranoside and /? -nitrophenyl- «-/ ->-galactopyranoside, respectively, were used as synthetic substrates. The extracellular α-galactosidase activity was identified by evaluating the dye-zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6-bromo-2-naphthyl-α-D-galactopyranoside, hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish-brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α-galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α-galactosidase.