AN ALPHA-GLUCOSIDASE FROM PERIMICROVILLAR MEMBRANES OF DYSDERCUS-PERUVIANUS (HEMIPTERA, PYRRHOCORIDAE) MIDGUT CELLS - PURIFICATION AND PROPERTIES

Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) has a major alpha-gluc osidase bound to perimicrovillar membranes, which are the lipoprotein membranes ensheathing the midgut cell microvillar membranes in hemipte rans. The enzyme was solubilized in detergent and purified to homogene ity by means of affinity chromatography on Concanavalin A-Sepharose, i on-exchange on Mono Q and preparative polyacrylamide gel electrophores is (PAGE). The yield, purification factor and final specific activity were, respectively: 3.5%, 50-fold, 7.6 U/mg of protein. The alpha-gluc osidase is a glycoprotein with a pH optimum of 5.0. M(r) values were 6 1,000 (SDS-PAGE), 120,000 (gel filtration), 130,000 (ultracentrifugati on), or 431,000 (electrophoresis in native conditions). The data sugge st that the alpha-glucosidase occurs in vivo as dimers and, during ele ctrophoresis, as octamers. Taking into account k(cat)/K-m ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltode xtrins up to maltotetraose. Using p-substituted phenyl alpha-glucoside s as substrates, it was shown that aglycone leaving is rate limiting a nd that the reaction constant rho is negative, suggesting the intermed iary formation of a carbonium ion in the reaction path. Erythritol, de lta-gluconolactone and Tris are simple linear competitive inhibitors o f the alpha-glucosidase. Experiments involving competition between sub strates suggested that the enzyme operates in accordance with rapid-eq uilibrium kinetics, hydrolyzing maltose, p-nitrophenyl-alpha-D-glucosi de, sucrose and turanose at the same active site. Evidence suggests th at the active site may have five sub-sites.