| Abstract
| - Ca ions inhibit microtubule (MT) assembly and induce disassembly of the reconstituted MT. This Ca sensitivity of the MT assembly system varies markedly with the assembly medium. The half-maximal inhibitions of MT reassembly from crude extract (CE) of porcine brain and from purified microtubular proteins (PMP) occurred at free Ca2+ concentrations of 10 μM and 700 μM, respectively, in the presence of a low Mg concentration (0.5 mM) and at low ionic strength (10 mM MES and 50 mM KCl) (Nishida, E. & Sakai, H. (1977) J. Biochem. 82, 303-306). Increase in the Mg concentration from 0.5 mM to 5 mM decreased the Ca2+ concentrations required for the half-maximal inhibition of CE and PMP from 10 to 2 μM and from 700 to about 50 (mM, respectively. Upon increasing the KC1 concentration from 50 to 200 mM, the Ca2+ concentration for the half-maximal inhibition of PMP decreased to 10 μM from 700 μM. Concentrations of KC1 higher than 200 mM inhibited MT assembly by suppressing the initiation process. However, the addition of outer fiber fragments permitted growth of MT. This MT elongation was also inhibited by 10 μM Ca2+. Moreover, the MT growth induced by mixing purified tubulin dimers with outer fiber fragments showed the same Ca sensitivity as MT assembly from PMP. RNA decreased the initial rate of MT assembly from PMP, as reported by others. Addition of Ca2+ caused the initial rate to increase, although the final extent of MT assembly was suppressed. However, the Ca sensitivity as determined by the plateau level of the viscosity increase was not significantly altered in the presence of RNA. High concentrations of Na glutamate as well as glycerol augmented and stabilized the assembled MTs and lowered the Ca sensitivity.
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