Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica.
An alpha-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-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-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 MUalphaGlc 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.
Bravo-Torres
José C
JC
Facultad de Química, Instituto de Investigación en Biología Experimental, Universidad de Guanajuato, Apartado Postal No 187, Guanajuato, Gto 36000, México.
Villagómez-Castro
Julio C
JC
Calvo-Méndez
Carlos
C
Flores-Carreón
Arturo
A
López-Romero
Everardo
E
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004748:Entamoeba histolytica / Q000201:enzymology*; D000520:alpha-Glucosidases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism
D000520:alpha-Glucosidases