4mu α d glucopyranoside

Acid alpha‐glucosidase enzyme activity was assayed by 4MU α‐D‐glucopyranoside (Sigma–Aldrich) as previously described.¹⁶ Protein concentration was measured using a BCA protein assay kit (Thermo Scientific). Briefly, cells were extracted with distilled water. Cell lysates were diluted with .2 mol/L phosphate‐citrate buffer (pH 4.3) and incubated with 6 mmol/L 4MU α‐D‐glucopyranoside and 100 µmol/L acarbose (Sigma–Aldrich) at 37°C for 30 min followed by the addition of .15 mol/L glycine carbonate buffer (pH 10.4). Fluorescence intensity in samples was measured using a fluorometer RF‐XXX (Shimadzu Corporation). Enzyme activity was expressed as nmol/mg protein/h.

GAA enzyme activity was measured as described previously.^{11 (link)} Briefly, cells were homogenized in 200 µl distilled water and the supernatant was collected after centrifugation at 14,000×g for 10 minutes at 4°C. Protein concentration of the supernatant was measured by the bicinchoninic acid assay (BCA) protein assay. Then 80 µl homogenate containing 15 µg of protein and 120 µl 4-MU buffer containing 100 µl of 6mmol/l 4MU-α-D-glucopyranoside (Sigma Aldrich) and 20 µl of 100 µmol/l Acarbose (Sigma Aldrich) were incubated at 37°C for 30 minutes. After 3.8 ml of 0.15 mol/l glycine carbonate stop buffer (pH 10.4) was added, fluorescent intensity was measured using a spectrophotometer and enzyme activity (nmol/hr/mg protein) was calculated according to the intensity and protein concentration.