Nickel (Ni 2+) enhancement of microtubule assembly in vitrois dependent on GTP function

Abstract

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni 2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni 2+ damaging actions on MT, we have focused on dissecting the role of the Ni 2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A 350 at 27°C using purified bovine brain MT proteins containing 162 μM each of Mg 2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 μM, respectively. Replacement of GTP (25–87%) with increasing [NiCl 2] while keeping the sum of [GTP] and [Ni 2+] constant at 500 μM enabled MT assembly to proceed with shortened “lags” but reaching the same maximum plateau levels or elongation rates as with 500 μM GTP only. However, in reactions with Ni 2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni 2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca 2+ disruption than those assembled with 500 μM GTP only. Notably, in the presence of 500 μM Ni 2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni 2+ replacements of GTP. These results point to an important role of GTP/Ni 2+ interaction in modulating the Ni 2+ enhancement of MT assembly in vitro.