Purification and characterization of bovine brain glucocerebrosidase.
Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52 degrees C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-beta-epoxide, an irreversible inhibitor, and gluconolactone, a competitive inhibitor.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D005942:Gluconates / Q000378:metabolism; D005959:Glucosidases / Q000302:isolation & purification*; D005962:Glucosylceramidase / Q000302:isolation & purification* / Q000378:metabolism; D005963:Glucosylceramides / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D007783:Lactones; D008970:Molecular Weight
D005942:Gluconates; D005963:Glucosylceramides; D007783:Lactones; D006923:Hymecromone; C034888:4-methylumbelliferyl glucuronide; D005959:Glucosidases; D005962:Glucosylceramidase; C010730:beta-glucono-1,5-lactone