Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain.
The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.
D000079:Acetaldehyde / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002417:Cattle; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D010446:Peptide Fragments; D014404:Tubulin; D012710:Serum Albumin, Bovine; D000079:Acetaldehyde