Biology Reference
In-Depth Information
19.2.3.1
Notes
The solvent used to develop the column depends on which compounds are being
analyzed and their polarity. Preliminary test runs with different percentages of meth-
anol/water need to be carried out to work out the optimal solvent and elution time
of each compound. For example, when running ZMP and docetaxel, 70% (v/v)
methanol/water works well, and the column is developed in the same buffer with
no gradient. When ZMP, docetaxel, and peloruside are run, the samples are dissolved
in (v/v) 55% methanol/water and the column developed with three isocratic steps of
13 min 55% methanol/water, 10 min 70% methanol/water, and 10 min 55% metha-
nol/water. The absorbance of each compound is followed at its respectivewavelength.
Ligand in the supernatant is unbound, whereas that in the pellet is bound. If the test
ligand is in the pellet fraction, it is occupying its binding site. In the case of a cova-
lently reacting ligand, the compound will disappear from the supernatant but will not
be seen in the pellet as the covalently bound ligand cannot be extracted. For a test
ligand that binds reversibly, displacement of the ligand from its site with excess con-
trol ligand results in the test ligand only being present in the supernatant, and this in-
dicates the test ligand and the competitor ligand share the same site (
Fig. 19.2
B).
If no competition is detected by this second experiment and both test ligand and
laulimalide/peloruside are detected simultaneously bound to the MT, it means that a
new binding site is likely to be present.
19.2.4
Stoichiometry
Once it is known that a compound binds to MTs, it is important to determine the stoi-
chiometry in which it binds to its binding site. Currently, all known taxoid site and
laulimalide/peloruside site compounds bind to tubulin in a 1:1 stoichiometry, with
one ligand per heterodimer (
D´az & Andreu, 1993; Pera et al., 2010
). To confirm
the test ligand is reacting in the same way, the stoichiometry needs to be investigated.
1.
Measure the stoichiometry of MSA binding to cross-linked MTs by incubating
10
M taxoid binding sites in stabilized MTs with increasing amounts of
ligand (0-50
m
M) in GAB-0.1 mM GTP buffer. Incubate a sample with no
tubulin in the same way with 10
m
M ligand.
2.
Separate MTs with their bound MSA from unbound MSA by ultracentrifugation
(20 min, 25
C). Collect the supernatant and resuspend the pellet in 10 mM
NaPi buffer pH 7.0.
3.
Add an internal standard to the samples. Extract the ligands from the samples
with three volumes of dichloromethane, dry, and resuspend the sample in
solvent (
Section 2.3.1
). Analyze the samples by HPLC as described in
Section 2.3
, steps 5 and 6.
m
If the stoichiometry is 1:1, then given that there are 10
M binding sites, the amount
of ligand in the pellet should increase until it plateaus at 10
m
M, at which concen-
tration the binding sites are saturated. The ligand concentration in the supernatant
should then begin to increase as more ligand is added (
Fig. 19.3
A).
m
