CYTOSOLIC SIALIDASE FROM PIG BRAIN - A PROTEIN COMPLEX CONTAINING CATALYTIC AND PROTECTIVE UNITS

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three ban ds using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved in to a catalytic unit (M(r) 30 kDa), active but very labile 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 uni t. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a(1) a s substrate, the catalytic unit showed a Michaelis-Menten kinetics, an d the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten ki netics was exhibited with MU-NeuAc in both cases. The apparent V-max a nd K-m values of the catalytic unit for MU-NeuAc and GD1a were 105.1 a nd 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 p rotective units and 2-3 catalytic units. The sialidase complex and pro tective units did not display any beta-D-galactosidase, beta-D-N-acety l-glucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxy peptidase activities.