Purification and Characterization of Multiple Forms of α-Galactosidase in Cucumis melo Plants

Abstract

Summary Four distinct enzyme forms of α-galactosidase (α-D-galactoside galactohydrolase, E.C. 3.2.1.22) were resolved from Cucumis melo L. plants. The enzymes were purified using gel filtration, affinity chromatography and native PAGE, and have been characterized. Three acid forms with pH optima of 6.0, 5.5 and 6.2, and one alkaline form with a pH optimum of 7.5 were identified. The molecular mass of these forms of α-galactosidase ranged between 35 and 70 kD. Acid α-galactosidase III seems to be a dimer with a M R of 16.5 kD. None of the α-galactosidases were glycosylated. Both acid and alkaline forms were inhibited by galactose as well as by high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPG). The K M values for the substrates stachyose, raffinose, melibiose and manninotriose were different. One of the acid α-galactosidases showed higher affinity for stachyose than for raffinose, while the two others preferred raffinose as substrate. The alkaline α-galactosidase revealed a similar affinity for stachyose and raffinose. The occurrence of these isoenzymes in organs of C. melo with different activities, depending on their development, indicates the metabolization of oligosaccharides by both acid and alkaline α-galactosidase forms. Our results suggest that multiple forms of α-galactosidase are required for the complete cleavage of the galactosyl moieties from galactosyl-oligosaccharides in C. melo plants.