Abstract
The assembly of pure tubulin into microtubules is induced by Mn(II), and the polymerized product displays the characteristic sensitivity of normal microtubules to low temperature. The interaction of Mn(II) with tubulin purified by phosphocellulose chromatography to remove microtubule-associated proteins was investigated using electron paramagnetic resonance and atomic absorption spectroscopy. Mn(II) interacts with tubulin at about one high affinity site with an apparent dissociation constant (KD) of 1.6 +/- 0.3 microM and at 8 +/- 2 low affinity (KD = 0.38 +/- 0.18 mM) sites. The binding of Mn(II) to tubulin at the high affinity site was accompanied by release of tubulin-bound Mg(II) from the protein. Thus, Mn(II) substitutes for Mg(II) at the high affinity site and in promoting the assembly of tubulin. Mg(II), Co(II), and Zn(II) can displace Mn(II) from manganese-tubulin, but Ca(II) cannot. The electron paramagnetic resonance spectrum at 9.1 GHz of Mn(II) bound to the high affinity divalent cation site of tubulin is characterized by broadened hyperfine structure and the absence of solid state spectral features, suggesting that the bulk solvent is accessible to the bound metal ion.
Highlights
The assembly ofpure tubulin into microtubules is EXPERIMENTALPROCEDURES inducedbyMn(II),and the polymerized product dis- Preparation of Tubulin-Tubulin fromfresh bovinebrainwas plays the characteristic sensitivity of normal microtu- prepared by amodification [9] of the recyclization procedure of bules to low temperature
The protein (10 to 15 mg/ml) was stored at with tubulin purified by phosphocellulose chromatography to remove microtubule-associated proteins was investigated using electron paramagnetic resonance and atomic absorption spectroscopy
Electrophoresis on SDSacrylamide gels indicated that the phosphocellulose-purified tubulin accompanied by release of tubulin-bound Mg(I1) from wasessentiallydevoid of lower molecularweight proteins
Summary
Of protein systems[8].The resultsof the present study estab- It was assumed for this analysis that Mn(I1) interactswith tubulin lish the feasibility of employing this metal ion as a paramag- a t two independent classes of binding sites. Ligand binding for this netic probeof the high affinity divalent cation binding site of case is described by Equation 1 [15]: the tubulin dimer. ’ Theabbreviations used are: Mes, 2-(N-morpholino)ethanesulfonic acid;EGTA, ethyleneglycol bis(P-aminoethyl ether)N,N,N’,N’tetraacetic acid; Pipes, 1,4-piperazinediethanesulfonic acid; M e S O , dimethyl sulfoxide; SDS, sodium dodecyl sulfate
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