Abstract
Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI approximately 7.0 suggesting the involvement of histidyl residues. Modification of histidyl residues of tubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/total His residues) for 3 min at 25 degreesC completely inhibited vinblastine-induced polymerization with little effect on microtubule assembly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Demodification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [14C]DEPC localized both of these histidyl residues on beta-tubulin at beta227 and beta264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time approximately 4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on alpha-tubulin at alpha88. Thus, two charged histidyl residues are obligatorily involved in vinblastine-induced polymerization, whereas a different histidyl residue on a different tubulin monomer is involved in microtubule assembly.
Highlights
Diethylpyrocarbonate (DEPC)1 on vinblastine-induced spiral formation, and we have compared these to changes produced in microtubule assembly
Demodification of the derivatized tubulin by hydroxylamine was carried out as follows: tubulin (5.3 mg/ml) was reacted with 1 mM DEPC for 4 min at 25 °C, and the reaction was quenched with 3 mM imidazole
Vinblastine has multiple effects on the properties of tubulin as a function of its concentration. At substoichiometric concentrations it reduces the dynamic instability of microtubules [12]; at intermediate concentrations it blocks assembly from tubulin dimers [13, 14]; and at higher concentrations it promotes the formation of various non-microtubule polymers whose structures are a function of the solvent composition
Summary
Diethylpyrocarbonate (DEPC) on vinblastine-induced spiral formation, and we have compared these to changes produced in microtubule assembly. Because the rate of histidyl reaction toward DEPC varies widely between and within proteins [3,4,5,6,7], such an approach would be useful only if the critical residues were the most reactive ones. This proved to be the case, and two highly reactive histidyl residues were found to be required for spiral formation, whereas 1–2 additional histidyl residues must be modified to block microtubule assembly
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