We have examined the effects of a number of organic anions, which stabilize tubulin, on tubulin polymerization, associated GTP hydrolysis, and polymer morphology. While microtubule-associated proteins, as well as glycerol, induced formation of typical microtubules in a reaction coupled to GTP hydrolysis at an initial 1:1 stoichiometry, the organic anions had varying effects. Only 2-(N-morpholino)ethanesulfonate induced formation of structures with the morphology of microtubules. With glutamate, fructose 1,6-bisphosphate, piperazine-N-N'-bis(2-ethanesulfonate), glutarate, and glucose 1-phosphate, the predominant structures formed were sheets of parallel protofilaments rather than microtubules. Creatine phosphate induced the formation of clusters of rings. GTP hydrolysis was closely coupled to polymerization only with glutamate. With creatine phosphate, there was minimal GTP hydrolysis. With all other organic anions, GTP hydrolysis substantially exceeded polymerization at all time points, with the onset of hydrolysis significantly preceding the onset of turbidity development. Nevertheless, the rate of GTP hydrolysis was a sigmoidal function of tubulin concentration under all conditions examined, suggesting that tubulin-tubulin interactions are required for hydrolysis. All anion-induced reactions were temperature dependent and cold reversible, but only the creatine phosphate induced reaction was not inhibited by GDP, CA2+, or colchicine and did not require GTP.

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4-Piperazineethanesulfonate (Pipes) and 4-morpholineethanesulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature-dependent, cold-reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes-induced polymerization ; and turbidity was much more intense in the Pipes-induced than in the Mes-induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth-walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes.When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both low and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations.Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M in several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.The polymerization of tubulin has been studied in many laboratories under a variety of experimental conditions (for a review, see [l]). Most studies have been performed with microtubule protein, preparations prepared by assemblydisassembly [Z] and containing tubuliii and a variable number of microtubule-associated proteins (MAPs) [3,4]. There have, however, been an increasing number of reports describing agents which induce purified tubulin to polymerize. These include glycerol and high concentrations of Mg2+ [5], DEAEdextran [6], dimethylsulfoxide [7], high concentrations of Mg2+ alone [8], and glutamate [9].Himes and his colleagues also reported a relatively sluggish polymerization reaction with 0.4 M Pipes if MAP-free tubulin was used [lo], and that sulfonate buffers at high concentrations potentiated the polymerizing effect of dimethylsulfoxide [7]. In the course of our laboratory's examination of the effects of organic acids on tubulin stability and polymerization [9,11], we had observed brisk polymerization of highly purified tubulin in 0.8 M Pipes requiring no other component except GTP. Attempts to extend this observation to Mes were unsuccessful until we noted that the Mes-induced reaction required significantly higher concentrations of tubulin...