Table 2. Enzymatic assays description.
Enzyme | Substrates | Concentration (mM) | Reaction buffers | Substrate specificity |
β-glucocerebrosidase | 4-methylumbelliferyl-β-D-glucopyranoside | 10.0 | citrate/phosphate, pH 5.0, 0.2% sodium taurodeoxicolate | 100% |
α-mannosidase | 4-methylumbelliferyl-α-D-mannopyranoside | 3.0 | 0.2 M sodium acetate, pH 4.5 | 95% lysosomal α-mannosidase (5% neutral α-mannosidase) |
β-mannosidase | 4-methylumbelliferyl-β-D-mannopyranoside | 3.0 | citrate/phosphate, pH 4.5 | 100% |
β-hexosaminidase | 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside | 3.0 | citrate/phosphate, pH 4.5 | 100% |
β-galactosidase | 4-methylumbelliferyl-β-D-galactoside | 3.0 | citrate/phosphate, pH 4.5 | 100% |
α-fucosidase | 4-methylumbelliferyl-α-D-fucoside | 3.0 | citrate/phosphate, pH 4.5 | 100% |
cathepsin D and cathepsin E | MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys (Dnp)-D-Arg-NH2 | 0.03 | 50 mM sodium acetate, pH 4.0 (w or w/o 15 µM pepstatin A) | cathepsin D activity was determined subtracting the value obtained in the presence of Pepstatin A (cathepsin E activity) from the value in absence of inhibitor (cathepsin D + cathepsin E) |
The specific substrate and reaction conditions are reported for each analyzed lysosomal enzyme. Sample volumes, temperature and time of incubation are indicated in Material and Methods.