Abstract
It has been reported that highly reactive oxygen free radicals may play a crucial role in sarcolemmal injury following ischemia/reperfusion, and cause irreversible inhibition of Na+,K+-ATPase activity. To determine whether the ATP binding site of the cardiac Na+K+-ATPase is directly Involved in oxygen radical injury, purified rat cardiac Na+,K+-ATPase was exposed to the hydroxyl radical (OH) generated from 80 μM Fe3+-NTA and l mM H2O2 under various conditions. Exposure of Na+,K+-ATPase to OH for 20 min at 25 °C caused a 79% decrease in Na+,K+-ATPase activity. SDS-gel electrophoresis indicated that OH did not damage the primary structure of the enzyme. When Na+,K+-ATPase was premixed with 1 mM ATP before exposure to -OH, complete protection was observed: there was no loss of enzymatic activity. No inhibition was seen when purified Na+,K+-ATPase was exposed to 1 mM H2O2 or 80 μM Fe3+-NTA by themselves. EPR spectroscopy demonstrated that ATP did not scavenge OH. These results suggest that: 1} OH denatures the cardiac sarcolemmal Na+,K+-ATPase by directly attacking the ATP binding site; 2) occupation of the ATP binding site of the enzyme protects against -OH induced loss of enzymatic activity, and 3) the depletion of ATP that occurs during ischemia may enhance the toxic effect of OH formed at the time of reperfusion.
Original language | English (US) |
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Journal | FASEB Journal |
Volume | 10 |
Issue number | 3 |
State | Published - 1996 |
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Cell Biology