Purification and characterization of GM1 ganglioside beta-galactosidase from normal feline liver and brain.
GM1 ganglioside beta-galactosidase (GM1-beta-galactosidase) was purified from normal cat brain and liver by a combination of classical and affinity procedures. The final preparation of brain GM1-beta-galactosidase was enriched over 2000-fold with a 36% yield. However, the product was shown to contain several components by disc gel electrophoresis. GM1-beta-galactosidase was also purified from liver with greater than a 30 000-fold enrichment and 40% yield. The liver enzyme was judged homogeneous by disc gel electrophoresis at pH 4.3, 8.1, and 8.9 and by gel chromatography. Both liver and brain GM1-beta-galactosidase(s) eluted as sharp symmetrical peaks from Sephadex G-200 with molecular weights of 250 000 +/- 50 000. The apparent Km determined for 4-methylumbelliferyl beta-D-galactopyranoside (4-MU-Gal) using partially purified brain GM1-beta-galactosidase was 1.73 X 10(-4) M. Liver GM1-beta-galactosidase gave a Km with 4-MU-Gal of 3.25 X 10(-4) M and for [3H]GM1 ganglioside a Km of 4.51 X 10(-4) M was calculated. The pH optima of brain and liver GM1-beta-galactosidase using 4-MU-Gal was 3.8-4.5. By contrast, liver GM1-beta-galactosidase gave a sharp activity peak at pH 4.2 with [3H]GM1 ganglioside. Inhibition by mercuric chloride and sensitivity to hydrogen peroxide and persulfate suggest the involvement of a sulfhydryl in catalysis.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002415:Cats; D005696:Galactosidases / Q000378:metabolism*; D005732:Gangliosides; D005733:Gangliosidoses / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009928:Organ Specificity; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*
D005732:Gangliosides; D005696:Galactosidases; D001616:beta-Galactosidase