title: Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units.
abstract: Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.
authors.affiliation: Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Venerando
authors.forename: A
authors.initials: A
authors.lastname: Fiorilli
authors.forename: L
authors.initials: L
authors.lastname: Di Francesco
authors.forename: A
authors.initials: A
authors.lastname: Chiarini
authors.forename: E
authors.initials: E
authors.lastname: Monti
authors.forename: D
authors.initials: D
authors.lastname: Zizioli
authors.forename: G
authors.initials: G
authors.lastname: Tettamanti
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology*; D004795:Enzyme Stability; D007700:Kinetics; D009439:Neuraminidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
chemical_list: D009439:Neuraminidase