Acetaldehyde is known to form covalent adducts with tubulin and to inhibit microtubule formation. Available evidence indicates that lysine residues are prominently involved in adduct formation. Previous work has shown that lysines on tubulin can be divided into two general classes based upon their reactivity toward acetaldehyde; those of normal reactivity ("bulk" lysines) and a highly reactive lysine (HRL) located on the a-polypeptide subunit. We took advantage of the fact that the HRL is unreactive when tubulin is in the microtubule form to differentiate the effects of bulk from HRL adducts on tubulin polymerization. Under conditions where both bulk lysines and HRL formed adducts, 0.2 mol acetaldehyde/mol tubulin caused complete inhibition of polymerization. When we modified bulk lysines, but not HRL, tubulin polymerized essentially normally. Finally, when we first blocked bulk lysines on microtubules (HRL unreactive) using unlabeled acetaldehyde and then measured the amount of ["4Clacetaldehyde adduct formed with tubulin after depolymerization (HRL reactive), 0.08 mol acetaldehyde/mol tubulin resulted in completely impaired polymerization. These data show that microtubule formation is very sensitive to even small mole fractions of acetaldehydemodified tubulin (especially with HRL) and further suggest that small amounts of acetaldehyde adduct could be damaging to cytoskeleton function in the cell.

Acetaldehyde covalently binds to purified tubulin in vitro to form both stable and unstable adducts. The formation of stable adducts can be greatly facilitated by the inclusion of the relatively gentle and Schiff base specific reducing agent, sodium cyanoborohydride. Although the tubulin molecule has multiple lysine resides available to react with acetaldehyde, certain key lysine residues on the alpha-chain appear to be selective targets for adduct formation. The formation of alpha-chain specific stable acetaldehyde-tubulin adducts results in functional impairment of the ability of tubulin to polymerize. Under relatively physiologic conditions where acetaldehyde-to-protein ratios are low, alpha-chain specific binding is prominent. These results, coupled with the studies presented in another report in this volume, raise the possibility that low levels of adduct formation may be detrimental to the structure or function of certain proteins (e.g. tubulin) in the liver. The alteration of this or other biologically important proteins by sustained low levels of adduct formation may contribute to the pathogenesis of alcoholic liver injury.

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Ethanol, acetaldehyde, and acetate were investigated for their effects on bovine neurotubulin polymerization. Ethanol at concentrations as high as 50 mM did not affect the rate or extent of tubulin polymerization. Acetaldehyde inhibited tubulin polymerization in a concentration-dependent manner, with complete inhibition at 10 mM and slight inhibition at 1 mM. Sodium acetate caused a concentration-dependent increase in tubulin polymerization, however sodium chloride showed a similar effect. These data indicate that ethanol and acetate were not inhibitors of microtubule formation while acetaldehyde exhibited weak inhibitory activity.

The covalent binding of [14C]acetaldehyde to purified rabbit skeletal muscle actin was characterized. As we have found for other cytoskeletal proteins, actin formed stable covalent adducts under reductive and non-reductive conditions. Under non-reductive conditions, individual and competition binding studies versus albumin both showed that the G-form of actin is more reactive toward acetaldehyde than the F-form. When proteins were compared on an 'equi-lysine' basis under non-reducing conditions, G-actin was found to preferentially compete with albumin for binding to acetaldehyde. Time-course dialysis studies indicated that acetaldehyde-actin adducts become more stable with prolonged incubation at 37 degrees C. These data raise the possibility that actin could be a preferential target for adduct formation in cellular systems and will serve as the basis for ongoing studies aimed at defining the role of acetaldehyde-protein adducts in ethanol-induced cell injury.