LOCALIZATION OF CRITICAL HISTIDYL RESIDUES REQUIRED FOR VINBLASTINE-INDUCED TUBULIN POLYMERIZATION AND FOR MICROTUBULE ASSEMBLY

Vinblastine-induced tubulin polymerization is electrostatically regula ted and shows pH dependence with a pI similar to 7.0 suggesting the in volvement of histidyl residues. Modification of histidyl residues of t ubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/ total His residues) for 3 min at 25 degrees C completely inhibited vin blastine-induced polymerization with little effect on microtubule asse mbly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Dem odification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [C-14]DEPC localized both of these histidyl residues on beta-tub ulin at beta 227 and beta 264. Similarly, tubulin modification with DE PC for longer times (8 min) resulted in complete inhibition of microtu bule assembly, at which time similar to 4 histidyl residues had been m odified. This inhibition by DEPC was also reversed by hydroxylamine. T he third histidyl residue was found on alpha-tubulin at alpha 88. Thus , two charged histidyl residues are obligatorily involved in vinblasti ne-induced polymerization, whereas a different histidyl residue on a d ifferent tubulin monomer is involved in microtubule assembly.