Purification and biochemical characterization of a soluble alpha-glucosidase from the parasite Entamoeba histolytica.
A soluble alpha-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using a number of substrates indicated a preferential hydrolysis of isomaltose (alpha1,6) with much less activity on nigerose (alpha1,3) and maltose (alpha1,4). Trehalose (alpha1,1), kojibiose (alpha1,2) and cellobiose (beta1,4) were not cleaved at all. As expected, isomaltose competed away hydrolysis of 4-methylumbelliferyl-alpha-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable K(i) values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the alpha-glucosidase inhibitors 1-deoxynojirimycin, castanospermine and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited isomaltose hydrolysis in a competitive manner with K(i) values of 1.2 and 1.5 muM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably involved in glycogen metabolism.
Bravo-Torres
José C
JC
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Guanajuato, Gto. 36000, México.
Calvo-Méndez
Carlos
C
Flores-Carreón
Arturo
A
López-Romero
Everardo
E
D000818:Animals; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005936:Glucans / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism; D021122:Protein Subunits; D012995:Solubility; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000302:isolation & purification* / Q000378:metabolism*
D004187:Disaccharides; D004791:Enzyme Inhibitors; D005936:Glucans; D006023:Glycoproteins; D009005:Monosaccharides; D021122:Protein Subunits; C014637:laminaribiose; C008237:kojibiose; D000520:alpha-Glucosidases