Characterization of symbiosis specific forms of alpha-glucosidase from Glycine max root nodules

Abstract

Summary Three forms of α-glucosidase (EC 3.2.1.20) were isolated from soybean ( Glycine max L. Merr. cv. Maple Arrow) root nodules and two of them were further purified by a procedure including fractionation with PEG-8,000 followed by Q-Sepharose, S-Sepharose, Chromatofocusing and Superose 6 column chromatography. Chromatofocusing of the plant soluble fraction yielded two different isoforms, which were designated as I and II. Their isoelectric points were 7.3 (I) and 6.5 (II) as determined by native isoelectric focusing. The α-glucosidase I was further purified 502-fold to a specific activity of 1.8 units (mgprotein) -1 . In the peribacteroid space only one form, designated III, was resolved by chromatofocusing. The isoelectric point was 7.2 estimated by native isoelectric focusing. The α-glucosidase III was purified 86-fold to a specific activity of 3.1 units (mg protein) -1 . The α-glucosidases I and II had similar properties. The pH-optima were 5.2 (I) and 4.8 (III). The subunit molecular weights for α-glucosidase I and α-glucosidase III were estimated by SDS-PAGE to be 65 kDa and 66 kDa, respectively. Both enzymes hydrolyzed maltose, maltotriose, isomaltose and dextrin. Starch was only weakly hydrolyzed and neither a-glucosidase acted on sucrose. K m values for maltose were 0.91 mM (I) and 0.67 mM (III), and for PNPG 0.37 mM (I) and 0.76 mM (III). Ag + and Fe 2+ were the most potent inhibitors of both enzymes. The functions of these enzymes in the symbiotic compartments are discussed.