Purification and characterization of GM1 ganglioside β-galactosidase from normal feline liver and brain

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Abstract
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 MW 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 .times. 10-4 M. Liver GM1-.beta.-galactosidase gave a Km with 4-MU-Gal of 3.25 .times. 10-4 M and for [3H]GM1 ganglioside a Km of 4.51 .times. 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 HgCl2 and sensitivity to H2O2 and persulfate suggest the involvement of a sulfhydryl in catalysis.