The lectin nature and other characteristics of alpha-galactosidases from Vicia faba seeds

Abstract

Three forms of [alpha]-galactosidases (I, II

1 and II

2 with apparentmolecular weights of 160,000, 45,000 and 43,400 respectively), which had been reported earlier were purified from mature Vicia faba seeds. The purification procedure was improved by the use of a substrate-affinity step. All three forms displayed catalytic and hemagglutinin activities. Various experimental results suggested that the enzyme preparations were free from favin (apparent molecular weight 50,000), a lectin which also occurs in V.faba seeds. The sites for catalytic and lectin activities appeared to reside at separate loci in all forms of the enzyme, and the lectin activity was glucose/mannose specific. The use of equilibrium dialysis and spectrophotometric technique indicated the presence of 8, 4 and 2 lectin binding sites in [alpha]-galactosidases I, II

1 and II

2 respectively, and 4 catalytic sites in [alpha]-galactosidaseI. All three forms of the enzyme were glycoproteins, and in the case of [alpha]-galactosidase I a constituent glycopeptide was isolated and investigation of its structure by fast atom bombardment-mass spectrometry suggested that an asparagine-glucosamine linkage was present and that [alpha]-galactosidase I was a 'mannose-rich' type glycoprotein. [alpha]-Galactosidase I was found to be altered in structure upon incubation with endo-?-N-acetylglucosaminidase H (Endo H), which was possibly due to the removal of glycan chains. The effects of a range of concentrations of urea and methyl-[alpha]-D-mannoside on [alpha]-galactosidasel were examined. Conversion of this high molecular weight tetramer into enzymically active low molecular weight forms, possibly trimers, dimers and monomers, was achieved. The three forms of the enzyme were separated into further active forms on isoelectric focusing and chromate focusing.