4-Hydroxynonenal inhibits Na⁺-K⁺-ATPase

Hydroxynonenal binds rapidly to Na⁺-K⁺-ATPase, and this was accompanied by a decrease in measurable sulfhydryl groups and a loss of enzyme activity. The I₅₀ value for Na⁺-K⁺-ATPase inhibition by 4-hydroxynonenal was found to be 120 μM. Although the sulfhydryl groups could be completely restored with β-mercaptoethanol during the reaction of the Na⁺K⁺-ATPase-HNE- adduct, the Na⁺-K⁺-ATPase activity was only partially restored by this reducing agent. A combination of hydroxylamine and β-mercaptoethanol yielded the greatest recovery of enzyme activity, 85% of original. Thus, 4- hydroxynonenal binding to Na⁺-K⁺-ATPase led to an irreversible decrease of enzyme activity under the conditions employed. It is hypothesized that 4- hydroxynonenal reacts with sulfhydryls at sites on the enzyme that are inaccessible by β-mercaptoethanol. Furthermore, evidence was obtained that 4-hydroxynonenal reacts with other amino acids such as lysine to form adducts that also interfere with protein function.