The α-glucosidase
inhibitory activity assay was based on a standard fluorometric assay
as previously described63 (link) using human lysosomal
α-glucosidase-overexpressing preparations from transiently transfected
COS-7 cells.64 (link) Briefly, the inhibitor was
premixed with the appropriate amount of α-glucosidase-overexpressing
homogenates (10–30 μg of protein) and incubated for 10
min at 37 °C. Except in the cases ofαGA1 , AGT2 , AGT9 , and AGT11 , which were
dissolved in water, the reactions contained 3.5% dimethyl sulfoxide
from the inhibitor solutions. Subsequently, 4-methyl-umbelliferyl
α-d -glucopyranoside (Sigma-Aldrich, St. Louis, MO)
(final concentration = 1.26 mM) in 0.1 M citrate-phosphate buffer,
pH 4.0, was added in an incubation mixture of 70 μL. The reaction
was incubated for 60 min at 37 °C and was terminated by adding
200 μL of 0.5 M sodium carbonate, pH 10.7, with 0.25 g/L Triton
X-100. The release of the product 4-methylumbelliferone was measured
at 365 nm excitation/450 nm emission. The control samples were prepared
without the inhibitor. The percent inhibition of enzyme activity was
calculated using the following formula where control activity
is the enzyme activity
under the same conditions but without the inhibitor.
The IC50 value is defined as the concentration of compound inhibiting
50% of α-glucosidase activity under the stated assay conditions.
inhibitory activity assay was based on a standard fluorometric assay
as previously described63 (link) using human lysosomal
α-glucosidase-overexpressing preparations from transiently transfected
COS-7 cells.64 (link) Briefly, the inhibitor was
premixed with the appropriate amount of α-glucosidase-overexpressing
homogenates (10–30 μg of protein) and incubated for 10
min at 37 °C. Except in the cases of
dissolved in water, the reactions contained 3.5% dimethyl sulfoxide
from the inhibitor solutions. Subsequently, 4-methyl-umbelliferyl
α-
(final concentration = 1.26 mM) in 0.1 M citrate-phosphate buffer,
pH 4.0, was added in an incubation mixture of 70 μL. The reaction
was incubated for 60 min at 37 °C and was terminated by adding
200 μL of 0.5 M sodium carbonate, pH 10.7, with 0.25 g/L Triton
X-100. The release of the product 4-methylumbelliferone was measured
at 365 nm excitation/450 nm emission. The control samples were prepared
without the inhibitor. The percent inhibition of enzyme activity was
calculated using the following formula where control activity
is the enzyme activity
under the same conditions but without the inhibitor.
The IC50 value is defined as the concentration of compound inhibiting
50% of α-glucosidase activity under the stated assay conditions.
