CO-FACTOR OPTIMIZATION OF KINESIN-DRIVEN MICROTUBULE GLIDING

E. Unger, K.J. Bohm and R. Stracke
Dept. Mol. Cytology, Inst. Mol. Biotechnology, Beutenbergstr. 11, D-07745 Jena, Germany

To optimize the conditions of kinesin-driven microtubule motility we have investigated the influence of the co-factors Mg²⁺ (including some physiologically relevant or chemically similar divalent metal ions), temperature, and ATP.

In the presence of 0.5 mM Mg²⁺ and 0.5 mM ATP at 25^oC microtubules were translocated, as widely known, at rates of about 0.6 mm/s by pig brain kinesin. Gliding stopped almost completely when Mg²⁺ was removed. After re-addition of Mg²⁺, motility was restored. Ca²⁺, and in a reduced degree also Sr²⁺ and Ba²⁺, are able to substitute for Mg²⁺. Under appropriate conditions the efficiency of Ca²⁺ was about 30% to 40% compared to that of Mg²⁺. For both Mg²⁺ and Ca²⁺ the maximum speed was found at a concentration of 2.5 mM to be 1.07 and 0.36 µm/s, respectively. If Zn²⁺ or Mn²⁺ substituted for Mg²⁺ an arrest of microtubule movement was observed. Under these conditions the microtubules still remained bound to the glass surface-attached kinesin. The motility could not be restored by the re-addition of Mg²⁺. The results confirm the hypothesis that the molecular mechanisms of force generation and kinesin binding to microtubules are differently regulated processes.

The temperature dependency of microtubule gliding was biphasically exponentially dependent on temperature between 4^oC and 37^oC, with a break point at 22^oC reflecting intramolecular changes. At the highest temperature investigated (45^oC) gliding still took place at a rate of 0.95 µm/s.

By measuring gliding activity in dependence on the ATP concentration we found a vmax of 0.78 µm/s and a Km of 0.053 mM (at 0.5 mM Mg²⁺ and 23^oC). By combining the optimal co-factor conditions the maximum speed was found to be 1.47 µm/s.

This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (FKZ: 0311369)