Biology Reference
In-Depth Information
12.1.2.2.2
GTPase assay
Tubulin is incubated with Gs
a
QL for 20 min at 37
C in PEM buffer. The reaction is
stopped with 5% cold perchloric acid followed by mixing (vortexing). Reactions are
extracted by incubating with charcoal-phosphate (5% Norit-A-activated charcoal in
50 mM Na
3
PO
4
, pH 3.0) for 15 min on ice with periodic vortexing followed by cen-
trifugation two times at 16,000
g
for 30 min to remove the charcoal. Charcoal ab-
sorbs GTP
32
and leaves free Pi
32
in the supernatant. The supernatant is counted in a
scintillation counter (we use a Beckman LS6000I), and the tubulin GTPase activity is
expressed as millimole
32
P
i
released/min/mol tubulin. The result, shown in
Fig. 12.4
A, reveals that Gs
a
increases tubulin GTPase activity in a concentration-
dependent manner with EC50
20 mmol of GTP hydrolyzed/min/
mol of tubulin. Using this method, we also have demonstrated that Gs
a
-peptide
(270-284) corresponding to the interface between Gs
a
and tubulin also increases tu-
bulin GTPase activity in a concentration-dependent manner with
V
max
¼
¼
1.2
m
M,
V
max
¼
0.070 pmol
P
i
released/min/
m
g tubulin and EC50
¼
24
m
M(
Dave et al., 2011
).
12.1.3
Functional consequences of Gs
a
-mediated activation of
tubulin GTPase: Gs
a
and Gs
a
-derived peptides increase
microtubule dynamics
Microtubules are stabilized by the presence of GTP at the plus-end. As a result of
Gs
a
-activating tubulin GTPase, Gs
a
also increases microtubule dynamic instability.
We determine the ability of microtubule to polymerize in the presence of Gs
a
using a
microtubule polymerization assay and evaluate microtubule dynamics using real-
time measurement by video microscopy. A flowchart demonstrating the microtubule
assays protocol is shown in
Fig. 12.3
.
12.1.3.1
Microtubule polymerization assay
It is hypothesized that, for a given population of tubulin, the percentage present as mi-
crotubules is diminished in the presence of Gs
a
or peptides corresponding to the Gs
a
tubulin binding site. To test this, microtubule polymer mass is determined using 15
m
M
tubulin in PEM buffer with 200
m
MGTP, pH 6.9 (G-PEM). Tubulin is polymerized for
1 h at 37
C, Gs
a
Q227L
(exchanged into G-PEM buffer using a Microcon spin concen-
trator), added to microtubules for 1 h at 37
C, and the microtubules are then separated
from soluble tubulin at 100,000
g
for1hat37
C (Beckman TL-100). The final re-
action volume, including Gs
a
,is20
m
l. The pelleted protein is resuspended in 20
m
lof
water at 4
C. Two microliters of each fraction is run on a 10% SDS-polyacrylamide
gel (125 V, 2 h), followed by Coomassie Blue staining, to determine the relative mass
of polymerized versus soluble tubulin (
Fig. 12.4
B).
12.1.3.2
Microtubule dynamics assay by video microscopy
To study the effect of Gs
a
on microtubule dynamic instability, purified bovine brain
tubulin (15
m
M) is assembled onto sea urchin (
Strongylocentrotus purpuratus
)axo-
nemes in PMEMbuffer in the presence of 2 mMGTP. The reactionmixture is incubated
