Biomedical Engineering Reference
In-Depth Information
consist of a bulky blocking group on the
N
-terminus followed by
a three to four amino acid residue peptides with a fluorophore,
usually amido-4-methylcoumarin (AMC), at the
C
-terminus.
The proteasome cleaves an amido bond between an amino acid
and the fluorophore, resulting in the release of a highly fluo-
rescent product that can be followed by fluorometry (
12
).
Depending on the amino acid sequence of the synthetic pep-
tide, the three proteasome activities can be distinguished.
Some of the most commonly used substrates are succinyl-Leu-
Leu-Val-Tyr-AMC (Suc-LLVY-amc) for chymotrypsin-like
activity,
N
-
t
-butyloxycarbonyl-Leu-Ser-Thr-Arg-AMC (Boc-
LSTR-amc) for trypsin-like activity, and
N
-benzyloxycarbonyl-
Leu-Leu-Glu-AMC (Z-LLE-amc) for caspase-like activity,
respectively (
10
).
The different peptidase activities of the proteasome are opti-
mal at neutral to weakly alkaline pH values depending on the
substrates, but buffers at pH 7.5-8.0 work well for assaying pro-
teasome activities (
13
). When the proteasome activity is deter-
mined in crude homogenates, and since the substrates are not
100% specific for the proteasome, other intracellular proteases
may cleave the peptides giving overestimated results. Therefore,
the non-specific proteolysis is commonly determined in the pres-
ence of a proteasome inhibitor, such as lactacystin or MG132,
although care has to be taken in the interpretation of the results,
since other proteases might be also inhibited by these reagents
(
11, 13
).
To measure the ATP-dependent proteasome activity (26S
proteasome), crude homogenates and proteasome activity have
to be performed in the presence of ATP and 10-20% glycerol or
0.25 mM sucrose to isolate integral 26S proteasome and to stabi-
lize the complex, respectively (
12, 14
). In addition, KCl is also
needed to further decrease the contribution of the 20S protea-
some to peptide cleavage by suppressing its spontaneous activa-
tion (
14
). It was demonstrated that different concentrations of
ATP in the assay method affect differentially the proteasome
activity, and, therefore, for each case, the concentration of ATP
for optimal determination of 26S proteasome activity should be
calculated (
14
). A detailed method for measuring 26S protea-
some in purified proteasome fractions and in crude homogenates
can be found in (
12
).
A detailed protocol for measuring different activities of the
20S proteasome in a microplate reader fluorometer using fluoro-
genic substrates is presented. This protocol is modified from
combining protocols from other papers, and from our own
experience (
15-17
).
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