Purification and biochemical characterisation of a membrane-bound α-glucosidase from the parasite Entamoeba histolytica

Abstract

An α-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-α- d-glucopyranoside (MUαGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 °C. Although the enzyme preferentially hydrolysed nigerose (α1,3-linked), it also cleaved kojibiose (α1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (α1,4), trehalose (α1,1) and isomaltose (α1,6). Activity on α1,3- and α1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy- d-glucose and 6-deoxy- d-glucose were strong inhibitors of activity, whereas 2-deoxy- d-glucose and other monosaccharides had no effect. Enzyme activity on MUαGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)- N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.