The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu 21 , Cd 21 , Hg 21 and CrO 2± 4 inhibit the tubulin polymerization at an IC 50 of 14±24 mm 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 mm. BIAcore measurements for tubulin±tubulin interaction suggest that the presence of Cu 21 affects neither k off nor k on , but the amount of the bound tubulin. While the inhibitory effect of Cu 21 on tubulin polymerization is partially abolished by crosslinking 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 4 ) has the ability to penetrate all tissues in the body and produces oxidative stress and oxidative DNA damage by generating active oxygen radicals and reactive Cr(Vc) species [3].The assembly/disassembly process of MTs built up of tubulin dimers has been observed in intact cells, cell-free preparations, and reconstituted systems. The assembly and function of MTs are regulated by MAPs and a variety of other effectors and environmental conditions. Sulfhydryl reagents block tubulin polymerization in vitro [4] by reacting with two of the eight SH-groups per tubulin dimer [5] that are essential for MT assembly [6]. An early study indicated the interaction of sulfhydryl groups of tubulin with Cu 21 , Cd 21 , and Hg 21 [7]. Copper also affects cells' energy production by decreasing the glycolytic rate in the brain cytosolic fraction [8] and also in the muscle cytosolic fraction [9].Recently, we provided evidence for the combined enhancement of MT-assembly and glucose metabolism, which resulted in decreased sensitivity to copper toxicity [10]. The mutual enhancement effect appeared to be specific for neuronal systems as it was observed in microtubular protein-free cytosolic fractions (MPFCFs) from whole brain and from cultured neuroblastoma cells but not from muscle cytosolic fraction. Here we have extended our studies to other bivalent ions, Cu 21 , Cd 21 , CrO 2± 4 , Hg 21 , and Pb 21 , as well as the monovalent anion NO ± 2 . We characterized the binding of the ions to target proteins as well as its structural and functional consequences. These studies at different organization levels rendered it possible to establish a role for macromolecular associations in response to different stress effects.

Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregatesandbundlesthetubulesinasaltconcentrationdependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo. Microtubules (MT)1 constitute a crucial part of the cytoskeleton and are involved in a variety of cell functions such as maintenance of shape, organization of intracellular transport, motility, and cell division. The polymerization dynamics of MT is under strict control (1). In addition to small molecules (inorganic ions, guanine nucleotides, and drugs), numerous proteins are known to interact with MT as positive regulators of MT assembly (MAPs) either by promoting the polymerization of tubulin or by stabilizing MT (1, 2). Several other proteins including Op18/stathmin, katanin, and some kinesin-like proteins act as destabilizers (1, 2).As shown by various methods including co-sedimentation assays and immunostaining, many glycolytic enzymes can interact transiently with the actin and/or microtubular network either in vitro or in vivo; the properties of associated partners (e.g. the stability of cytoskeleton and the activity of the enzyme) are deeply influenced by the mutual interactions (Refs. 3-5; for review see Ref. 6). Recently, we found that two of the glycolytic enzymes, the M1 isoform of pyruvate kinase (PK) and Dictyostelium discoideum phosphofructokinase, can act as microtubule-destabilizing factors by inhibiting paclitaxel-induced polymerization of tubulin and by promoting disassembly of microtubules into thread-like oligomers (7,8).In the present study we have further characterized the PKtubulin/MT interaction with purified proteins using surface plasmon resonance technology, and we present data on the heterologous association of PK with MTs in brain extract, the protein composition of which approximates that of the living cells. We present evidence on the crucial role of acidic Cterminal fragments of MTs in the PK binding. We show the modulating role of phosphoenolpyruvate (PEP) on tubulin-PK as well as on MT-PK interaction at di...