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.