pH-Dependent Association-Dissociation of GM1-β-Galactosidase Purified from Porcine Spleen

Publisher Website
Google Scholar
Abstract
A β-galactosidase [EC 3.1.23] catalyzing the hydrolysis of GM1-ganglioside was purified from porcine spleen to a homogeneous form. By applying a hydrophobic chromatography procedure as the first purification step, the enzyme could be purified through subsequent purification steps as a dissociated form. The purified enzyme was a monomer with an apparent molecular weight of 70,000-74,000 at neutral pH and associated to a dimer with an apparent molecular weight of 158,000-160,000 at acidic pH, near optimal for its activity (pH 4.6). It had specific activities of 1,820 μmol/mg/h towards GM1 with an apparent Km of 3.18 × 10−5M, 1,880 μmol/mg/h towards lactosylceramide with an apparent Km of 1.99 × 10−4M, and 1,340 μmol/mg/h towards p-nitrophenyl-β-galactopyranoside (pNp-β-galactoside) with an apparent Km of 2.14 X10−4M. Kinetic studies suggested that common catalytic site(s) cleaved the two natural substrates mentioned above.