title: Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition.
abstract: In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: B S
authors.initials: BS
authors.lastname: Levison
authors.forename: J
authors.initials: J
authors.lastname: Wiemels
authors.forename: J
authors.initials: J
authors.lastname: Szasz
authors.forename: H
authors.initials: H
authors.lastname: Sternlicht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000493:pharmacokinetics / Q000494:pharmacology*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D008969:Molecular Sequence Data; D009842:Oligopeptides; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators
chemical_list: D005561:Formates; D009842:Oligopeptides; D014404:Tubulin; D050257:Tubulin Modulators; D004047:Diethyl Pyrocarbonate