Characteristic features of an alpha-galactosidase from mung beans.
Two molecular forms, I and II (high and low molecular mass) of alpha-galactosidase were demonstrated in dry mung beans and a multi-step procedure was developed for isolating the tetrameric enzyme I in good yield. Two affinity chromatographic techniques were employed and an overall 10 000-fold purification was achieved. The enzyme was able to catalyse the hydrolysis of alpha-galactosidic linkages as well as agglutinate rabbit erythrocytes (clot formation). The clot was temporary and dissolved on longer incubation, yielding free galactose. The pH optima for both activities were similar. The enzyme also destroyed human-blood-group-B activity and increased H activity. The effect of pH on Km and Vmcx of the enzyme indicated the importance of carboxyl (pK approximately equal to 4.0) and histidine (pK approximately equal to 6.5) groups for activity. This was confirmed by amino acid modification experiments in the absence and presence of the substrate. The stoichiometry of enzyme inactivation showed the probable presence of 12 carboxyl groups and 9 histidine imidazole groups/molecule enzyme in the active site. The effects of modification of the groups on enzymic and hemagglutinating activities were parallel. A model explaining the display of both the activities by the enzyme, is presented in which hemagglutination is shown to be due to the formation of slightly stable enzyme-substrate complex. It is proposed that a true lectin should not alter the covalent status of the binding sugar.
D000596:Amino Acids; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D005696:Galactosidases / Q000302:isolation & purification*; D006801:Humans; D037102:Lectins / Q000302:isolation & purification; D037121:Plant Lectins; D010946:Plants, Medicinal; D011487:Protein Conformation; D011817:Rabbits; D000519:alpha-Galactosidase / Q000302:isolation & purification* / Q000494:pharmacology
D000596:Amino Acids; D037102:Lectins; D037121:Plant Lectins; D005696:Galactosidases; D000519:alpha-Galactosidase