TITLE: Lysosomal GCase (glucocerebrosidase) activity assay
AUTHORS: Sara Gomes, Esther Sammler

[DESCRIPTION]
Here we report a method to measure enzyme activity of lysosomal glucocerebrosidase (GBA1, GCase) by monitoring the hydrolysis of the fluorescent substrate 4-methylumbelliferyl-β-D-glucopyranoside. The assay is performed at low pH, at which non-lysosomal glucocerebrosidase activity is expected to be low. This is consistent with the abolishment of 4-MUG hydrolysis in the presence of the GBA1 inhibitor CBE. Our data shows that GBA1 activity is significantly increased in purified lysosomes compared to the whole cell extract.

[STEPS]
SECTION: Buffer preparation
1. 0.1M citric acid: dissolve 19.2 g  in 1 L

SECTION: Buffer preparation
2. 0.2M sodium phosphate: dissolve 28.4 g in1 L.

SECTION: Buffer preparation
3. Citrate-phosphate buffer, pH 5.4: mix 44.2 mLwith 56.8 mL to make 100mL citrate-phosphate buffer, pH 5.4.

SECTION: Buffer preparation
4. 0.5M EDTA: dissolve 20.8 g in 80 mL Adjust to pH 8and top up to 100 mL.

SECTION: Buffer preparation
5. Assay buffer: to make 500 mL assay buffer, add 1.25 g , 5 g, and 1 mL to 500 mL.

SECTION: Buffer preparation
6. Stop buffer: dissolve 37.5 g  in 400 mL. Adjust to pH 12.5 and top-up volume to 500 mL.

SECTION: Buffer preparation
7. 10mM 4-MU calibrator stock solution: dissolve 17.6 mg in 10 mL. Aliquot and store at -20 °C, protected from light.

SECTION: Substrate preparation
9. Dissolve 4.2 mg in 2.5 mL (final concentration = 5 millimolar (mM)). A sonicator water bath may be used to facilitate dissolution.
 




SECTION: Sample preparation
10. Add 5 µg of protein from whole cell extracts or 1 µg of protein from Lyso-IP samples into the wells of a flat bottom black 96-well plate in duplicate.
 



SECTION: Sample preparation
11. Top up volume to 80 µL with assay buffer.

SECTION: Sample preparation
12. Add 1.2 µL of DMSO or 25mM CBE to each sample well.

SECTION: Sample preparation
13. Prepare blank samples in duplicate: add 80 µL to two empty wells.

SECTION: Sample preparation
14. Prepare calibrator wells: designate 24 empty wells for the calibrators and add 100 µL assay buffer to each of these wells.

SECTION: Enzymatic reaction
15. Add 20 µL of the 5mM 4-MUG solution prepared in step 9 to each of the sample and blank wells.

SECTION: Enzymatic reaction
16. Cover the plate and incubate at 37 °Cfor 60 min.

 

SECTION: Preparation of calibrator serial dilutions
17. During the incubation, thaw an aliquot of 10mM 4-MU.
SECTION: Preparation of calibrator serial dilutions
18. Label 11 1.5mL microcentrifuge tubes with numbers 1-11.
SECTION: Preparation of calibrator serial dilutions
19. Add 1 mL to tube 1.

SECTION: Preparation of calibrator serial dilutions
20. Add 500 µLto tubes 2-11.

SECTION: Preparation of calibrator serial dilutions
21. Add 2 µL to tube 1.

SECTION: Preparation of calibrator serial dilutions
22. Mix by pipetting up-and-down and transfer 500 µL from tube 1 to tube 2.

SECTION: Preparation of calibrator serial dilutions
23. Repeat step 22 sequentially for the remaining tubes. At the end, only tube 11 should contain 1 mL.
 
 
 

SECTION: Stop reaction and fluorescence measurement
24. Add 100 µL to each sample and blank well.

SECTION: Stop reaction and fluorescence measurement
25. Add 100 µLto 2 of the calibrator wells. These will be the blanks for the calibration curve.

SECTION: Stop reaction and fluorescence measurement
26. Add 100 µL of each calibrator solution prepared in step 23 to 2 of the calibrator wells.

SECTION: Stop reaction and fluorescence measurement
27. Measure fluorescence intensity in a plate reader (Ex/Em = 350/460).
 

SECTION: Data analysis
28. Plot the fluorescence intensity of the calibrator against the corresponding amounts
of 4-MU in picomoles (pmol). Determine the linear equation representing this
relationship.
SECTION: Data analysis
29. Using the calibration curve equation, estimate the amount of released 4-MU in picomoles for the
samples.
SECTION: Data analysis
30. Divide the estimated amount of released 4-MU by the amount of protein lysate in milligrams (mg) and the incubation time in minutes. This yields the GCase activity, expressed in terms of released 4-MU in
picomoles per milligram per minute (pmol/mg/min).


 

SECTION: Buffer preparation
8. 25 mM CBE: dissolve 5 mg in 1.23 mL. Aliquot and store at -20°C.