HYDROLYSIS OF FUCOSYL-GM1 GANGLIOSIDE BY PURIFIED PELLET-ASSOCIATED HUMAN BRAIN AND HUMAN LIVER ALPHA-L-FUCOSIDASES WITHOUT ACTIVATOR PROTEINS OR DETERGENTS

Pellet-associated human brain α-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290000-fold purification, a 58% yield and a final specific activity of 11500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent K(m) for the synthetic substrate 4-methylumbelliferyl α-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver α-L-fucosidases were both capable of hydrolysing fucosyl-G(M1) ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37°C was found at pH 3.4 for both α-L-fucosidases, with no activity at pH values above 4.0.