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 labile upon storage at 4°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 1 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 V max and K m values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2.10 −5 and 1.6.10 −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 β- d-galactosidase, β- d -N- acetylglucosaminidase , α- l-fucosidase, α- d-glucosidase and carboxypeptidase activities.