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Introduction Set-Up Results Conclusions

 

Mobility of Taxol in Microtubule Bundles

Jennifer L. Ross1 and D. Kuchnir Fygenson1,2

1Physics Dept. & 2BMSE Pgm. University of California, Santa Barbara

 

 

 

ABSTRACT    Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching (FRAP) on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With > 63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, koff. It was found that 10 koffGMPCPP ~ koffGTP, consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With < 63% sites occupied, recovery times decreased as ~[TAX]-1/5 for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within ~7 nm of each other.

 

  

 

Microtubules are:

 


Tubulin dimers have binding sites for nucleotides,
proteins, and therapeutic drugs.

 



Alberts, et al. Molecular Biology of the Cell (1994)

 

   

 
 

 

 

Binding and Mobility of Taxol:
 

 

Binding is a reversible, first order reaction:

[TAX] + [dimer] [TAX-dimer]



The equilibrium dissociation constant, KD, is found by:

KD = koff / kon = [TAX][dimer] / [TAX-dimer]

KDGMPCPP=15 nM; KDGTP=3300 nM

 

Mobility of Taxol in Microtubules is Affected by:

1. Steric hindrance (bumping) and pores (escape)

 

 

2. Presence of binding sites
 


A. Density of binding sites

 
 

i. Varied by changing [TAX]

 
    ii. fill ratio =
[TAX]/KD
(1+[TAX]/KD)
 

 




high [TAX], fill ratio 1

 




low [TAX], fill ratio 0



More open binding sites create more potential barriers to movement.
Diffusion will be faster when the [TAX] increases.

 

B.Affinity of binding sites
 


i. Affinity ~ KD-1
ii. KDGMPCPP = 15 nM (higher affinity)
iii. KDGTP = 3300 nM


 
   

 

 

Introduction Set-Up Results Conclusions

 

 

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