Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 181

3. For 2 wells of each sample, pipette 200 ml of AB using a
repeating pipette. To the other well for the same sample,
pipette 200 ml of IB.
4. Incubate at 37°C for 10 min in the fluorometer (see Note 9).
5. Meanwhile, prepare the substrate solution to the proteasome
activity that will be measured (see substrate solutions in
Subheading 2.2 ).
6. After pre-incubation, take the plate from the fluorometer and
using a repeating pipette, add 25 ml of the chosen substrate
solution (e.g., Suc-LLVY-amc substrate solution for measur-
ing the chymotrypsin-like activity).
7. Mix the plate for 5-10 s using the built-in automixing feature
of the microplate reader fluorometer and read the fluores-
cence intensity along the time using ex 380nm /em 460nm (AMC)
in the fluorometer for around 20 min in 1 min intervals
at 37°C.
8. Take the slope (reaction kinetic) obtained from each sample
with and without the proteasome inhibitor.

3.3. Calculation
of the Proteasome
Activity

To express any peptidase activity of the proteasome, the slopes of
the linear kinetics of the reaction are taken by using the instru-
ment software. The slope obtained in the replicate measured in
the presence of MG132 is subtracted to the average of the other
two replicates to eliminate the proteasome non-specific activity.
This value can then be expressed as percentage of the sample con-
sidered as the control of the experiment.
Instead of the relative activity between the samples, the results
can be expressed as specific activity of the enzyme by using the
fluorescence intensity (FI) of one AMC standard obtained in the
same final volume of the assay and the following equation:
Specific activity [nmol AMC/min/mg] = [slope (FI/
min) × AMC standard concentration (mM) × volume of assay (ml)]/
[fluorescence of AMC standard (FI) × quantity of protein (mg)].
An example is given for understanding how proteasome spe-
cific activity is calculated in an assay performed with 10 mg of
protein and in a final volume of 230 ml:

1. Calculate proteasome activity:
1.1. Slope of activity measured without MG132: 22 FI/min
1.2. Slope of activity measured with MG132: 2 FI/min
1.3. Proteasome activity will be: 22-2 = 20 FI/min
2. Measure fluorescence of an AMC standard:
2.1. Fluorescence of 5 mM AMC standard: 330 FI
2.2. Fluorescence of blank: 30 FI
2.3. AMC standard fluorescence will be: 330-30 = 300 FI