Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition.
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.
Levison
B S
BS
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.
Wiemels
J
J
Szasz
J
J
Sternlicht
H
H
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
D005561:Formates; D009842:Oligopeptides; D014404:Tubulin; D050257:Tubulin Modulators; D004047:Diethyl Pyrocarbonate