|Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic Rate - BioAssay Summary
Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. It is a lysosomal hydrolase that is required for the degradation of a small percentage (1-3%) of cellular glycogen in human. Deficiency of this enzyme results in glycogen-storage disease type II (GSDII), also referred to as Pompe disease, an autosomal more ..
BioActive Compound: 1
Depositor Specified Assays
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: 1R03MH084841-01
Assay Submitter (PI): Wei Zheng
NCGC Assay Overview:
Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. It is a lysosomal hydrolase that is required for the degradation of a small percentage (1-3%) of cellular glycogen in human. Deficiency of this enzyme results in glycogen-storage disease type II (GSDII), also referred to as Pompe disease, an autosomal recessive disorder. Structurally normal glycogen is accumulated in lysosomes and cytoplasm in affected patients, primarily in muscle tissues. Excessive glycogen storage within lysosomes may interrupt normal functioning of other organelles and leads to cellular injury. In turn, this leads to enlargement and dysfunction of the entire organ involved (eg, cardiomyopathy and muscle weakness).
It has reported that the improper folding and trafficking of alpha-glucosidase resulting from the genetic mutations may account for a significant number of Pompe patients. N-butyl-deoxynojirimycin, an inhibitor of alpha-glucosidase, was reported to exhibit the pharmacological chaperone activity, which significantly increases the mutant enzyme activity in cells. We optimized this alpha-glucosidase assay in 1536-well plate format for identifying the novel small molecule inhibitors with the structures other than the sugar analogs in order to develop the new pharmacological chaperones.
This assay was performed to establish that the compounds tested increase substrate turnover by directly following the consumption of substrate and formation of product using LC-MS.
The catalysis rate of the 4MU-alpha-Gluc substrate by acid alpha-glucosidase was measured using LC-MS. Various concentrations of compound were added to enzyme solution in tubes. This was incubated with substrate and then stopped with stop solution. LC-MS was performed on each solution to determine the amount of product. The formation of product showed a dose-dependent increase.
(1) Add 50 ul of enzyme to 1.5 ml tubes (4 nM final)
(2) Add 0.5 ul compounds in DMSO solution. The final titration was 0.7 nM to 77 uM.
(3) Add 50 ul of substrate (400 uM final)
(4) Incubate at room temperature for 20 min.
(5) Add 50 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)
(6) Run LC-MS and integrate peaks to quantitate amount of product
** Test Concentration.
Data Table (Concise)