4-Hydroxynonenal inhibits Na+-K+-ATPase

Werner G. Siems, Sharon J. Hapner, Frederik J.G.M. Van Kuijk

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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.

Original languageEnglish (US)
Pages (from-to)215-223
Number of pages9
JournalFree Radical Biology and Medicine
Issue number2
StatePublished - 1996

Bibliographical note

Funding Information:
Acknowledgements -- This work was supported by a grant from the National Eye Institute (EY 08818) and by the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under Grant Nos. 90-0327 and 93-NL036. The U.S. government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright notation hereon. This work was also supported in part by a Research to Prevent Blindness development grant. The authors thank Matt Rees for synthesis of 4-hydroxynonenal, which was used for the binding and inhibition experiments. The authors also thank Herman Swarts, Department of Biochemistry MF, University of Nijmegen, Netherlands, for assistance with the assay for measuring ATPase activity, and his helpful discussion and comments on this manuscript.


  • 4-Hydroxynonenal
  • Aldehydes
  • Free radicals
  • Lipid peroxidation
  • Na-K-ATPase
  • Sulfhydryl groups
  • β-Mercaptoethanol


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