Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena.
A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013769:Tetrahymena pyriformis / Q000201:enzymology* / Q000254:growth & development; D000520:alpha-Glucosidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D002241:Carbohydrates; D005959:Glucosidases; D000520:alpha-Glucosidases