title: Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta.
abstract: Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: G T
authors.initials: GT
authors.lastname: van der Horst
authors.forename: N J
authors.initials: NJ
authors.lastname: Galjart
authors.forename: A
authors.initials: A
authors.lastname: d'Azzo
authors.forename: H
authors.initials: H
authors.lastname: Galjaard
authors.forename: F W
authors.initials: FW
authors.lastname: Verheijen
mesh_terms: D002846:Chromatography, Affinity; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D009439:Neuraminidase / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D009439:Neuraminidase; D001616:beta-Galactosidase