Biology Reference
In-Depth Information
19.2.1.3
Transmission electron microscopy
Once the MT-stabilizing activity of a test compound has been confirmed, it is im-
portant to visually confirm that the formed polymers are MTs. The reason for this is
that some compounds induce tubulin aggregation rather than MT assembly, yet the
presence of aggregates increases light scattering and fluorescence similar to MTs.
To check for MT morphology, pipet the polymerized tubules onto 400-mesh
carbon- and Formvar-coated copper grids (or equivalent), stain with 2% phospho-
tungstate, and observe in a transmission electron microscope at 20,000
magnification.
If the above assays are positive for MSA activity, the next step in its character-
iza
tion is to determine its binding site. In principle, the best strategy to probe the
binding site is to perform various competition assays against compounds that bind
to the two known binding sites-the taxoid site, and the laulimalide/peloruside site.
19.2.2
Flutax displacement assay
A binding competition assay using stabilized taxoid binding sites and a fluorescent
derivative of paclitaxel that binds the taxoid site (Flutax-1 from Calbiochem
®
or
Flutax-2 purchased from us) can be performed to determine whether an MSA binds
to the taxoid site. This assay measures the binding affinity and kinetic binding con-
stant of a ligand in reference to its ability to displace the reference ligand from its
binding site.
The equilibrium binding constants of test ligands can be determined from anisot-
ropy titration measurements carried out at different temperatures by measuring fluo-
rescence intensities. The displacement isotherm of each ligand is determined in a
black 96-well plate (Nunc
®
FluoroNunc
™
, Sigma-Aldrich) in a fluorescence polar-
ization microplate reader. This protocol is described in
D
´
az and Buey (2007)
or is
available from the author. Equilibrium constants for Flutax-2 are presented in
Table 19.1
. Taxoid site ligands can be used as positive controls to compete with
Flutax-2. The displacement isotherm for the test ligand needs to be measured a
number of times at different temperatures in different plates.
A typical experiment is presented in
Fig. 19.2
A. A decrease in the anisotropy
indicates displacement of Flutax from the binding site. Docetaxel, ZMP, and
Table 19.1
Equilibrium constants of Flutax-2 binding to microtubules
26
C 7
C 0
C 2
C 5
C 7
C 0
C 2
C
K
a
(10
7
M
1
)
6.5
5.9
4.6
4.2
3.0
2.2
2.0
1.8
v
o
(Flutax-
2
bound
/Flutax-
2
total
)
0.378
0.563
0.522
0.508
0.451
0.398
0.382
0.364
