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J Biol Chem, Vol. 273, Issue 47, 31131-31137, November 20, 1998

Localization of Critical Histidyl Residues Required for Vinblastine-induced Tubulin Polymerization and for Microtubule Assembly

From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI ~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 °C 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 -tubulin at 227 and 264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time ~4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on -tubulin at 88. 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.

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