Glycosidases in seeds

Two forms of a-galactosidases (I and II) have been shown to exist in Vicia faba seeds and these have been purified 3660- and 337-fold, respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzyme I and II, as determined by gel filtration, were 209,000 and 38,000,respectively. The carbohydrate contents of enzyme I and II were 25.0% and 2.8%, respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive sub-units in the presence of 6M-urea. a-Galactosidases I and II showed different pH optima and K and V values with various natural and synthetic substrates and also differed in their thermal stabilities. The effect of temperature on various kinetic parameters has also been examined. Both enzymes are inhibited by excess substrate (p-nitrophenyl a-D-galactoside): with enzyme I this is competitive and is caused by the galactosyl moiety. Enzyme I is inhibited by various metal ions and by oligosaccharides possessing two terminal non-reducing galactose residues and to a lesser extentby L-arabinose and D-fucose. The effect of pH on K and V values suggests that carboxyl and imidazole (histidine) groups are involved in the catalytic activity of enzyme I. Photo-oxidation experiments with I also suggest that an imidazole group is present at the active site. Histochemical and tissue fractionation studies show that the enzyme is widely distributed in seedling tissues and is non-particulate. Aerobic conditions increase the level of a-galactosidase during germination and both giberellic acid aid kinetin enhance synthesis of the enzyme in vivo. As germination proceeds, the concentration of a-galactosyl sucrose derivatives decreases with a simultaneous decrease in the level of enayme I and an increase in that of II.