Characterization of a glycoprotein alpha-galactosidase from lentil seeds (Lens culinaris).
alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), an enzyme responsible for mobilizing the raffinose family of oligosaccharides in legume seeds, has been isolated from lentils (Lens culinaris) and purified about 4,000-fold. The Sephadex gel filtration profile showed the presence of two forms of the enzyme, alpha-galactosidase I with an apparent Mr = 160,000 and alpha-galactosidase II of Mr = 40,000. Enzyme II readily aggregates to form I when any attempt is made to concentrate the solution. Thus, only enzyme I was purified and its properties studied. The multistep purification procedure included affinity binding of the enzyme to concanavalin A-Sepharose, indicating its glycoprotein nature with glucose/mannose termini of the carbohydrate moieties. The amino acid and carbohydrate compositions of the native enzyme and that of the glycopeptide obtained from pronase-digested, denatured enzyme have been determined. Asparagine seems to be involved in forming the linkage with the carbohydrate moiety. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme I shows a single protein band with Mr = 40,000 which also stains with periodic acid-Schiff reagent. Thus, the enzyme consists of four identical glycoprotein subunits. The isoelectric point of the enzyme is 8.0. The pH optima of enzyme I and II are 6.1 and 4.7, respectively. The substrate specificity and the mode of substrate inhibition of enzyme I is discussed. The effect of temperature on Vmax and Km of the enzyme is presented; at pH 6.1 the energy of activation is 62.1 kJ/mol and the delta H value is -34.3 kJ/mol in the temperature range 20-50 degrees C. Preliminary studies show that enzyme I possesses hemagglutinating properties with glucose/mannose specificity.
D007887:Fabaceae / Q000201:enzymology*; D005696:Galactosidases / Q000378:metabolism*; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D010946:Plants, Medicinal; D012639:Seeds / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000378:metabolism*
D046911:Macromolecular Substances; D005696:Galactosidases; D000519:alpha-Galactosidase