Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae).
Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q. gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed.
Fonseca
Fábio V
FV
Polgenix Inc., 11000 Cedar Avenue, Suite 260, Cleveland, OH 44106, USA.
Silva
José R
JR
Samuels
Richard I
RI
DaMatta
Renato A
RA
Terra
Walter R
WR
Silva
Carlos P
CP
D000818:Animals; D002852:Chromatography, Ion Exchange; D004064:Digestive System / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D006430:Hemiptera / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008871:Microvilli / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism
D005960:Glucosides; D019476:Insect Proteins; C019502:4-nitrophenyl alpha-glucoside; D013395:Sucrose; D008320:Maltose