Tubulin unpolymerized

In favorable systems, the coherent movement of neuro-filaments and microtubule proteins provides strong evidence for the structural hypothesis. Striking evidence was provided by pulse-labeling experiments in which NF proteins moved over periods of weeks as a bell-shaped wave with little or no trailing of NF protein. Similarly, coordinated transport of tubulin and MAPs makes sense only if MTs are being moved, since MAPs do not interact with unpolymerized tubulin [31]. 

Microtubules in the cytoskeleton and mitotic apparatus are also in a state of dynamic equilibrium and flux with unpolymerized tubulin, and tubulin appears to be an excellent example of the proteins which Pauling (1953) postulated to exist as globular protomers or as insoluble, fibrous, supramolecular structures akin to unpolymerized and polymeric hemoglobin S. The current view of the microtubule cytoskeleton in nondividing celb comes from the development of tubulin-specific antibodies for indirect immunofluorescent localization of microtubules (Fuller et al., 1975 Weber et al., 1975). The general structural features of such cyto-... 

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

The structure based on solution NMR data reflects the influence of epothilone on the early steps of the tubulin assembly process, when the tubulin/EpoA complex is still in its soluble unpolymerized form and binding of EpoA is not as tight as with polymerized tubulin. The binding site of unpolymerized tubulin is expected to differ to a certain extent from that in polymerized MT. Still, similar to PTX, epothi-lones interact with the M loop and it is highly conceivable that the M loop is locked in a conformation already in the tubulin-dimer/epothilone complex that enhances lateral M loop contacts required for MT formation. 

Interestingly, the initial tubulin coordinates used in the study of HTI-286 are those of straight tubulin in 2D sheets (PDB entry 1JFF) [83], The X-ray structure of the T2R complex with vinblastine showed that the unpolymerized tubulin heterodimer is bent and that there are local conformational changes at the interdimer interface in comparison with straight tubulin [13], The discrepancy between the experimental [12] and computationally predicted [31] binding modes of colchicine has been attributed to the fact that the computational model was derived from... 

A series of small peptides were prepared in order to identify the minimal unit that could mimic the effect of the N-terminal cap domain. The structure of the most efficient (I19L, Fig. 35) was studied by NMR and molecular modeling [142]. The concentration dependence of inhibition of tubulin polymerization suggested that the peptides bind stoichiometrically to unpolymerized tubulin rather than to assembled MT. It is well known that the stability of the tubulin/stathmin complex decreases upon phosphorylation of stathmin [8]. In agreement with this, phosphorylated I19L was found to be fourfold less efficient than its unphosphorylated form. 

The mechanism of action of the vinca alkaloids is that of the inhibition of the polymerization of tubulin to microtubules. The cellular protein tubulin, which occurs in a- and /3-forms, is essential for proper cellular function. During mitosis tubulin polymerizes to form microtubules, which are long tube-shaped protein polymers. The equilibrium between unpolymerized a- and /3-tubulin and microtubules is an important one and any disruption of this equilibrium can send dividing cells into mitotic block and apoptosis. The vinca alkaloids bind to /3-tubulin at a different site from paclitaxel (Taxol) and act to prevent tubulin assembly. 

FIGURE 20-11 Dynamic instability model of microtubule growth and shrinkage. GTP-bound a(3-tubulin subunits (red) add preferentially to the (-t) end of a preexisting microtubule. After incorporation of a subunit, the GTP (red dot) bound to the (i-tubulin monomer is hydrolyzed to GDP Only microtubules whose (+) ends are associated with GTP-tubulin (those with a GTP cap) are stable and can serve as primers for the polymerization of additional tubulin. Microtubules with GDP-tubulin (blue) at the (+) end (those with a GDP cap) are rapidly depolymerized and may disappear within 1 minute. At high concentrations of unpolymerized GTP-tubulin, the rate of addition of tubulin is faster than the rate of hydrolysis of the GTP bound in the microtubule or the rate of dissociation of GTP-tubulin from microtubule ends thus the microtubule grows. At low concentrations of unpolymerized GTP-tubulin, the rate of addition of tubulin is decreased consequently, the rate of GTP hydrolysis exceeds the rate of addition of tubulin subunits and a GDP cap forms. Because the GDP cap is unstable, the microtubule end peels apart to release tubulin subunits. [See T Mitchison and M. Kirschner, 1984, Nature 312 237 ... 

Ankyrin, anchorin, syndein a membrane protein found in erythrocytes and brain, which binds Spectrin (see) to the membrane. Spectrin-binding activity was first isolated as a proteolytic fragment of M, 72,000 the M, of the entire protein is 215,000. There are about 10 copies of A. per erythrocyte ghost, i.e. the number required to bind all the spectrin dimers in a 1 1 complex. In the erythrocyte, A. links spectrin to the cytoplasmic part of Band 3 (a transmembrane protein), to the anion-transport protein, and to microtubules. A. is also able to bind unpolymerized tubulin. [V. Bennett, Annu. Rev. Biochem. 54 (1985) 273-304 V. Bennet Ankyrins adaptors between diverse plasma membrane proteins and the cytoplasm J. Biol. 

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