Biology Reference
In-Depth Information
Additional information may be obtained about the nature and shape of the poly-
mers produced in a polymerizing solution of tubulin by collecting the OD at multiple
wavelengths and determining the wavelength dependence of OD. This is often a
valuable procedure and has been described in several publications, but is beyond
the scope of this paper (see, e.g.,
Andreu & Timasheff, 1982; Detrich et al., 1985
).
We have determined the critical concentration,
C
C
, and the turbidity coefficient
e
*, for a number of buffer conditions, using a single preparation of purified tubulin
and a single temperature. These results are presented in
Table 14.1
for the conditions
shown in the curves in
Fig. 14.2
as well as other buffer conditions. These data can
provide part of the information for designing a polymerization experiment.
Several points should be made about the data in
Table 14.1
. It is clear that the crit-
ical concentration is strongly influenced by the buffer chosen for the polymerization. A
fairly high concentration of tubulin is required for assembly to occur in Pipes/Mg
buffer alone, though addition of 1 M glycerol lowers this by about twofold. Addition
of 1 M TMAO or 10
M paclitaxel lowers the required tubulin concentration by a fac-
tor of 10, as does adding 1 M glutamate (not shown). Use of 1 M glutamate alone has a
similar result. A slightly less obvious result of buffer choice is revealed by the turbidity
coefficient
m
e
*. Pipes/Mg buffer alone, or with 1 M glycerol or with 1 M TMAO, pro-
mote assembly that has a
0.23, consistent with polymers that are mostly MT.
1 M glutamate alone, or addition of 1 M glutamate to Pipes/Mg buffer (not shown) or
addition of 10
m
M paclitaxel to Pipes/Mg buffer, promotes assembly that has a
e
*of
0.5 or greater, indicating that many sheet polymers are also present in the solution.
A further point concerns the temperature. The values of
C
C
shown in
Table 14.1
were determined at 30
C. Note that in some cases, especially paclitaxel-promoted
assembly, increasing the temperature to 37
C is not likely to cause much increase in
polymer yield, because the
C
C
is already very low at 30
C.
e
*of
14.1.4
Polymerization promoters
Tubulinwill polymerize in a simple buffer that containsMg
2
þ
andGTP and is near neu-
tral pH. However, the
C
C
can be rather high (see
Table 14.1
), so it is not unusual to add
components to enhance polymerization by lowering the
C
C
. These fall into two catego-
ries: “tubulin-specific” and “thermodynamic.” Tubulin-specific agents require concen-
trations that are comparable to tubulin (tens of
M) and havemore-or-less specific sites
of interactions. Examples are paclitaxel (Taxol
™
) and proteins such as microtubule-
associated proteins (MAPs) or polyamines. Nontubulin-specific or “thermodynamic”
promoters require concentrations of hundreds of mM to 1 M or higher and favor poly-
merization by “molecular crowding” or water-exclusion effects. These are often natural
organic osmolytes and include glycerol, glutamate, and TMAO (
Hamel & Lin, 1981;
Lee & Timasheff, 1975; Sackett, 1997
), but also include dimethylsulfoxide (DMSO),
often used topromote polymerization and simultaneously to introduce sparingly soluble
test compounds (
Himes, Burton, &Gaito, 1977
). Glutamate is an unusual example of a
promoter, since it can be both the buffer and promoter—1 MNa glutamate is a complete
solution in which tubulin-GTP will polymerize.
m
