Purification and characterization of multiple forms of alpha-galactosidase in Cucumis melo plants

Purification and characterization of multiple forms of alpha-galactosidase in Cucumis melo plants

2000

A:PS

Four distinct enzyme forms of alpha-galactosidase (alpha-D-galactoside galactohydrolase, E.C. 3.2.1.22) were resolved from melon cv. Ranjadew 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 alpha-galactosidase ranged between 35 and 70 kDa. Acid alpha-galactosidase III seems to be a dimer with a MR of 16.5 kDa. None of the alpha-galactosidases were glycosylated. Both acid and alkaline forms were inhibited by galactose as well as by high concentrations of p-nitrophenyl-alpha-D-galactopyranoside (pNPG). The KM values for the substrates stachyose, raffinose, melibiose and manninotriose were different. One of the acid alpha-galactosidases showed higher affinity for stachyose than for raffinose, while the two others preferred raffinose as substrate. The alkaline alpha-galactosidase revealed a similar affinity for stachyose and raffinose. The occurrence of these isoenzymes in organs of melons with different activities, depending on their development, indicates the metabolization of oligosaccharides by both acid and alkaline alpha-galactosidase forms. Our results suggest that multiple forms of alpha-galactosidase are required for the complete cleavage of the galactosyl moieties from galactosyl-oligosaccharides in melon plants.

v.156(4):483-491, JOURNAL OF PLANT PHYSIOLOGY

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