Gaucher disease: Physical, kinetic and immunologic investigations of human and canine acid β-glucosidase

Kinetic and immunologic techniques were developed to investigate the nature of the acid β-glucosidase (β-Glc) defects which results in human and canine Gaucher disease (GD). Two new affinity columns, using the potent inhibitors of β-Glc (N-alkyl-deoxynojirimycins) as affinity ligands, were synthesized and methods were developed to obtain homogeneous β-Glc from normal human placenta. Polyclonal and monoclonal (representing 14 different epitopes from 18 clones) antibodies were produced to the pure normal β-Glc. Monospecific polyclonal IgG and tritiated-bromo-conduritol B epoxide ([3H]Br-CBE), a specific covalent active site directed inhibitor of β-Glc, were used to quantitate the functional catalytic sites in normal and Type 1 Ashkenazi Jewish GD (AJGD) enzyme preparations: The kcat values for several new substrates with the mutant enzymes from spleen were about 1.5-fold less than the respective normal enzyme, indicating a nearly normal catalytic capacity of the mutant enzymes. Immunoblotting studies with polyclonal or several monoclonal antibodies indicated three molecular forms of β-Glc (Mr = 67,000, 62,000 to 65,000 and 58,000) in fibroblast extracts from normals and Type 1 AJGD patients. In comparison, only one form of cross-reacting immunologic material (CRIM) was detected in fibroblast extracts from Types 2 and 3 or several non-Jewish Type 1 GD patients