Organization‐dependent effects of toxic bivalent ions

Abstract

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.

Surface plasmon resonance study of the actin-myosin sarcomeric complex and tubulin dimers

“…Gold film chips (CM5) coated with carboxymethylated dextran were obtained from of 50 µL to reach room temperature and start polymerizing, we are confident that in our measurements mainly free tubulin dimers were present and not MTs since (using spectrophotometry and monitoring the absorption curve) we had previously determined that the characteristic time for our tubulin to polymerize into MTs was of the order of 45 minutes at room temperature (data not shown) agreeing with literature [26].…”

Combined zebrafish-yeast chemical-genetic screens reveal gene–copper-nutrition interactions that modulate melanocyte pigmentation

“…Tubulins require post-translation folding, assembly into heterodimers, and then assembly into microtubules. In vitro and cell-based studies have shown that microtubules disassemble in excess copper (Liliom et al, 1999;Liliom et al, 2000;Nawaz et al, 2005;Pribyl et al, 2008;Liu et al, 2009). Copper might bind the sulfhydral groups of microtubules and thereby block microtubule assembly and/or induce disassembly (Wallin et al, 1977).…”

Towards Experimental Tests of Quantum Effects in Cytoskeletal Proteins

“…Our SPR measurements took place at 24 o C and the time between injection and measurement was of the order of 10 s. Measurements were taken for times up to 5 minutes. Tubulin does not polymerize at 0 o C and although 10sec is adequate time for our sample of 50 µL to reach room temperature and start polymerizing, we are confident that in our measurements mainly free tubulin dimers were present and not MTs since (using spectrophotometry and monitoring the absorption curve) we had previously determined that the characteristic time for our tubulin to polymerize into MTs was of the order of 45 minutes at room temperature (data not shown) agreeing with literature [140].…”