GM1 ganglioside beta-galactosidases from bovine liver.
Two GM1 ganglioside beta-galactosidases, multimeric form (enzyme I) and monomeric form (enzyme IV), have been purified from bovine liver by the procedures comprising Sephadex G-100 gel filtration, affinity chromatographies on Concanavalin A (Con A)-Sepharose and p-aminophenyl thio-beta-galactoside-CH-Sepharose (PATG-Sepharose) and Sephadex G-200 gel filtration. The multimeric form of the enzyme was purified 13,000-fold and monomeric form was 68,700-fold. On sodium dodecyl sulfate poly acrylamide gel electrophoresis, the monomeric form of the enzyme gave a single protein band with a molecular weight of 65,000, while the multimeric form gave two minor protein bands with molecular weights of 32,000 and 20,000 in addition to the major band at 65,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide. Enzyme I showed a broad pH optimum between pH 4.3 and 5.0, while enzyme IV was most active at pH 4.75. The pI values of beta-galactosidases I and IV were 4.6 and 5.8, respectively. Both enzymes were quite stable upon preincubation at 45 degrees C under acidic condition (pH 4.5), but rapidly lost their activities under neutral condition (pH 7.0). The apparent Km values for GM1 ganglioside of beta-galactosidases I and IV were calculated to be 2.0 x 10(-4) M and 3.3 x 10(-4) M, respectively.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005696:Galactosidases / Q000302:isolation & purification*; D005698:Galactosylceramidase; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000032:analysis / Q000302:isolation & purification*
D005696:Galactosidases; C021500:lactosylceramidase; D001616:beta-Galactosidase; D005698:Galactosylceramidase