Organization-dependent effects of toxic bivalent ions.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu²⁺, Cd²⁺, Hg²⁺ and CrO^2–₄ inhibit the tubulin polymerization at an IC₅₀ of 14–24 µm with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 µm. BIAcore measurements for tubulin–tubulin interaction suggest that the presence of Cu²⁺ affects neither k_off nor k_on, but the amount of the bound tubulin. While the inhibitory effect of Cu²⁺ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO₄^2–. No inhibitory effect of Cu²⁺ or CrO^2–₄ on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu²⁺ or CrO^2–₄ ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu²⁺, Cd²⁺, and Hg²⁺ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC₅₀ of 10–14 µm with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems. [ABSTRACT FROM AUTHOR]