ATP protects against free radical induced inhibition of the cardiac Na⁺,K⁺-ATPase

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.