Induction of Polymerization of Purified Tubulin by Sulfonate Buffers

Abstract

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...

The effect of MinC on FtsZ polymerization is pH dependent and can be counteracted by ZapA

“…S2). Because of these results, and the finding that the presence of Pipes itself can promote polymerization of the FtsZ homologue tubulin [27] the Pipes buffer was not used in further experiments.…”

Interactions of 2-Methoxyestradiol, an Endogenous Mammalian Metabolite, with Unpolymerized Tubulin and with Tubulin Polymers

“…It was feasible to explore these possibilities because (1) with glycerol tubulin will polymerize into microtubules both with (Shelanski et al, 1973) and without MAPs (Lee & Timasheff, 1975), and MAPs, when present, enter the polymer; and because (2) with high sulfonate buffer concentrations, Mes induces microtubule formation while Pipes induces formation of a mixture of sheets and microtubules, with sheets predominating (Waxman et al, 1981). a Reaction mixtures (0.4 mL) contained 15 µM (1.5 mg/mL) tubulin, 1.0 M monosodium glutamate (pH 6.6), 1.0 mM MgCl2, 0.1 mM GTP, and, if indicated "initially", [ 3 H]2ME.…”

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

“…In our own studies we have been routinely disappointed by low yields of MAPs obtained by chromatography of microtubule protein on DEAE-cellulose with subsequent heat treatment . In an effort to increase the amounts of MAPs we could obtain, we began to explore the selective polymerization of tubulin by high concentrations of organic anions (Waxman et al, 1981; as a means to separate tubulin and MAPs. Materials and Methods Materials. Mes, ATP, GTP, and GDP were obtained from including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent.…”