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 alpha-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 290,000-fold purification, a 58% yield and a final specific activity of 11,500 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 Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.
Hopfer
R L
RL
Department of Chemistry, Lehigh University, Bethlehem, PA 18015.
Johnson
S W
SW
Masserini
M
M
Giuliani
A
A
Alhadeff
J A
JA
D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D005644:alpha-L-Fucosidase / Q000302:isolation & purification / Q000378:metabolism*
D003902:Detergents; D005677:G(M1) Ganglioside; C039123:fucosyl GM1 ganglioside; D005644:alpha-L-Fucosidase