Purification and characterization of human liver β-galactosidase from a patient with the adult form of G_M1 gangliosidosis and a normal control

β-Galactosidases were purified to homogeneity from livers of a normal control and a patient with the adult form of G_M1 gangliosidosis. The purification was achieved by chromatography on DEAE-Sepharose fast flow, Con A-Sepharose, p-aminophenyl-1-thio-β-d-galactopyrarose, and QAE-Mono Q. The normal and mutant enzymes were purified about 5000-fold with a yield of 10% and 1800-fold with a yield of 34%, respectively, and could hydrolyze 4-methylumbelliferyl-β-d-galactoside, G_M1 ganglioside, and asialofetuin. The purified normal enzyme was eluted from a TSK gel G-4000SW column as three symmetrical peaks of protein which were coincident with the three peaks of enzyme activity. The enzyme in these three peaks had apparent molecular weights of 800 000 (polymer), 140 000 (dimer), and 65 000 (monomer), whereas the mutant enzyme was eluted as two symmetrical peaks of protein and enzyme activity. The apparent molecular weight of a major monomeric form of the enzyme (β-galactosidase A) was 60 000, and no dimeric form of the enzyme existed. Normal and mutant purified enzyme preparations migrated as a single major protein band with apparent molecular weights of 65 000 or 60 000, respectively, by SDS-polyacrylamide gel electrophoresis after treatment with mercaptoethanol. On isoelectric focussing, the mutant enzyme migrated more anodally than the normal enzyme. The mutant enzyme also had altered enzyme properties, such as pH optimum, K_m values, substrate specificity and heat-stability. These data on the characteristics of the purified enzyme preparations provide the first direct evidence that patients with the adult form of G_M1 gangliosidosis have a structurally altered β-galactosidase.