• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <0.1 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to release glucose from starch. The specific activity is >7,500 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Human embryonic kidney cell, HEK293-derived Ala70-Cys952, with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  99 kDa

• SDS-PAGE

  95-105 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 0.1 M Sodium Acetate, pH 4.5

• Recombinant Human Lysosomal alpha -Glucosidase (rhGAA) (Catalog # 8329-GH)

• Stop Solution: 4.4 mM Dinitrosalicylic Acid, 1 M Potassium Tartrate, 0.4 M Sodium Hydroxide in deionized water

• Standard: Maltose (Sigma, Catalog # M5885), 20 mM stock in deionized water

• Substrate: Starch from potato (Sigma, Catalog # 85642), 2% (w/v) stock in deionized water

• 96-well Clear Plate (Catalog # )

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 20 mM Maltose standard by adding 200 μL of the 20 mM Maltose Standard to 600 μL of Assay Buffer for a 5 mM stock. This is the first point of the standard curve.

2. Prepare the standard curve by performing five one-half serial dilutions of the 5 mM Maltose stock in Assay Buffer. Make sure there are 400 μL in each tube for each point of the curve (remove 400 μL from the last point of the curve). Prepare one tube with only 400 μL of Assay Buffer for the curve blank. The standard curve has a range of 19.5 to 625 nmol per well.

3. Dilute rhGAA to 32 ng/μL in Assay Buffer.

4. Dilute 2% starch to 1% in Assay Buffer by combining equal volumes of 2% starch and Assay Buffer.

5. Prepare reactions by combining 20 μL of diluted rhGAA with 380 μL of 1% starch (step 4). Include a control by combining 20 μL of Assay Buffer with 380 μL of 1% starch.

6. Vortex, spin, and then incubate reactions, control and standard curve at 37 °C for 1 hour.

7. Add 400 uL of Stop Solution to all vials, including standard curve.

8. Heat all vials at 95-100 °C for 6 minutes. Then, cool on ice. Tip: Use lid-locks on vials when heating.

9. Load 250 μL of each dilution of the standard curve, reactions and controls to empty wells in triplicate in plate.

10. Read plate at 546 nm (absorbance) in endpoint mode.

11. Calculate specific activity:

     *Derived from the maltose standard curve using linear or 4-parameter fitting and adjusted for curve blank.

Per Well:

• rhGAA: 0.200 μg

• Starch: 0.475%

Background: Lysosomal alpha-Glucosidase

Acid alpha-glucosidase (GAA) is an enzyme that is essential in the degradation of glycogen to glucose in the lysosome (1). Defects in GAA are the cause of glycogen storage disease II, also known as Pompe's disease, which is a rare autosomal recessive metabolic disorder that damages muscle and nerve cells throughout the body, primarily due to the accumulation of glycogen in the lysosome (2). Pompe disease occurs in babies, children, and adults who inherit a defective GAA gene and affects an estimated 5,000 to 10,000 people worldwide (3). Enzyme replacement therapy (ERT) is used to treat patients with this disease (4, 5).

• References:

1. Hoefsloot L.H. et al. (1988) EMBO J. 7:1697.

2. Wan, L. et al. (2008) J. Neurol. 255:831.

3. Fukuda, T. et al. (2007) Curr. Neurol. Neurosci. Rep. 7:71.

4. Van Gelder, C.M. et al. (2014) J Inherit Metab Dis. In press.

5. Toscano, A. and Schoser, B. (2013) J. Neurol. 260:951.

• Long Name:

  Glucosidase, Alpha; Acid

• Entrez Gene IDs:

  2548 (Human); 14387 (Mouse); 367562 (Rat)

• Alternate Names:

  Acid alpha-Glucosidase; Acid Maltase; Aglucosidase alfa; EC 3.2.1.20; GAA; glucosidase, alpha; acid; LYAG; Lysosomal alphaGlucosidase; Lysosomal alpha-Glucosidase