4-hydroxynonenal inhibits Na+-K+-ATPase

4-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 I-50 value for Na+-K+-ATPase inhibition by 4-hydroxynonenal was found to be 120 mu M. Although the sulfhydryl groups could be completely restored with P-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 beta-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 beta-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.