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. Author manuscript; available in PMC: 2010 Oct 25.
Published in final edited form as: Chem Biol Drug Des. 2009 Aug 18;74(4):358–368. doi: 10.1111/j.1747-0285.2009.00866.x

Figure 3. Inhibition of tubulin assembly in vitro.

Figure 3

Electrophoretically homogenous bovine brain tubulin (final concentration 10 μM; 1 mg/mL) was preincubated with test agents dissolved in DMSO (4% v/v final concentration) and monosodium glutamate (0.8 M final concentration) for 15 min at 30 °C. The reaction mixture was cooled to 0 °C and GTP (0.4 mM final concentration) was added. Reaction mixtures were transferred to cuvettes held at 2.5 °C in a temperature controlled multichannel spectrophotometer reading absorbance (turbidity) at 350 nm. Baselines were established and temperature was quickly raised to 30 °C (in approximately 1 min). After 20 min, the temperature was returned to 2.5 °C. (A) The known microtubule destabilizer, vinblastine (VBL) prevented tubulin assembly in a concentration-dependent manner; (B) (−)-Pironetin delayed the onset but increased the extent of tubulin assembly. (C) Effect of compound on tubulin assembly in the absence of GTP. Experimental conditions were as above except that to some samples no GTP was added. (D) Effect of (−)-pironetin on preformed microtubules. Instead of pre-incubation with test agent, drug in DMSO was added 7 min after initiating GTP-induced assembly.