THERMODYNAMIC ANALYSIS OF MICROTUBULE SELF-ASSEMBLY INVITRO

The temperature dependence of the equilibrium and the kinetics of microtubule assembly were investigated. Samples polymerized to equilibrium were analyzed by a quantitative sedimentation method (Johnson & Borisy, 1975). The kinetics of assembly were analyzed by monitoring changes in turbidity of samples induced to polymerize by temperature shift or by seeding with microtubule fragments (Johnson & Borisy, 1977). The results were consistent with a condensation polymerization mechanism although the system showed different behavior above and below 20 °C. Above 20 °C, a standard enthalpy of 8 kcal/mol and a standard entropy of 50 entropy units were obtained for the association of a 6 S tubulin subunit at the end of a microtubule. The apparent enthalpies of activation for the association and dissociation reactions were approximately 8 kcal/mol and 3 kcal/mol, respectively. The interpretation of these apparent thermodynamic quantities is discussed in terms of microtubule assembly as a steady-state balance of irreversible reactions coupled to the hydrolysis of GTP (Margolis & Wilson, 1978). Below 20 °C, the rate of association continued to decrease with an activation enthalpy of 8 kcal/mol. However, the rate of dissociation increased with decreasing temperature giving an apparent activation enthalpy of -30 kcal/mol and leading to a marked decrease in the equilibrium constant. The results below 20 °C were interpreted in terms of an additional reaction, possibly involving the association of microtubule-associated proteins at specific sites on the microtubule lattice. © 1979.