To study the rhGAA uptake and correction of GAA activity in PD fibroblasts, the cells were incubated with 50 µM rhGAA for 24 h, in the absence or in the presence of 10 mM L-CAR. Untreated cells were used for comparison. After the incubation, the cells were harvested by trypsinization and disrupted by 5 cycles of freezing and thawing.
GAA activity was assayed by using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU) (Sigma–Aldrich) according to a published procedure31 (link). Briefly, 25 µg of cell homogenates were incubated with the fluorogenic substrate (2 mM) in 0.2 M acetate buffer, pH 4.0, for 60 min in incubation mixtures of 100 µl. The reaction was stopped by adding 1 ml of glycine-carbonate buffer, 0.5 M, pH 10.7. Fluorescence was read at 365 nm (excitation) and 450 nm (emission) on a Promega GloMax Multidetection system fluorometer. Protein concentration in cell homogenates was measured by the Lowry assay.
GAA activity was assayed by using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU) (Sigma–Aldrich) according to a published procedure31 (link). Briefly, 25 µg of cell homogenates were incubated with the fluorogenic substrate (2 mM) in 0.2 M acetate buffer, pH 4.0, for 60 min in incubation mixtures of 100 µl. The reaction was stopped by adding 1 ml of glycine-carbonate buffer, 0.5 M, pH 10.7. Fluorescence was read at 365 nm (excitation) and 450 nm (emission) on a Promega GloMax Multidetection system fluorometer. Protein concentration in cell homogenates was measured by the Lowry assay.
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