pH-dependent association-dissociation of GM1-beta-galactosidase purified from porcine spleen

Abstract

A beta-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 mumol/mg/h towards GM1 with an apparent Km of 3.18 X 10(-5) M, 1,880 mumol/mg/h towards lactosylceramide with an apparent Km of 1.99 X 10(-4) M, and 1,340 mumol/mg/h towards p-nitrophenyl-beta-galactopyranoside (pNp-beta galactoside) with an apparent Km of 2,14 X 10(-4) M. Kinetic studies suggested that common catalytic site(s) cleaved the two natural substrates mentioned above.