title: Formation of ADP-sensitive phosphorylated intermediate in the electric eel Na, K-ATPase preparation.
abstract: The ADP-sensitive and K+ -sensitive phosphorylated forms of Na,K-ATPase (E1P and E2P, respectively) are believed to be the main phosphorylated intermediates of Na,K-ATPase. In the presence of 100 mM Na+, E2P is the major component of the phosphorylated form in all native Na,K-ATPase preparations known, including the microsomes of shark rectal glands. However, the Na,K-ATPase-rich membrane fragments of the electric eel formed a different type of phosphoenzyme, of which 80% was dephosphorylated by both 2 mM K+ and 1 mM ADP within 1 sec at 4 degrees under the same conditions. In the presence of oligomycin (50 micrograms/ml), only the percentage of K+ -sensitive phosphoenzyme was reduced in the eel enzyme, but no such effects were observed in the shark enzyme. However, the eel enzyme produced E2P-rich phosphoenzyme in the presence of 10 mM Na+, as did the shark enzyme in the presence of 100 mM Na+, and the shark enzyme formed the E1P-rich phosphoenzyme in the presence of 500-700 mM Na+, as did the eel enzyme in the presence of 100 mM Na+. These results suggest that the eel enzyme has a much higher affinity for Na+ on the Na+ site controlling the E1P-E2P equilibrium than does the shark enzyme, but we have not been able to explain this difference. Since the phosphorylated forms of both enzymes became more sensitive to both ADP and K+ at 25 degrees, both the interconversion of E1P to E2P and the reverse reaction seem to be accelerated by an increase in temperature. Oligomycin inhibited this conversion of E1P to E2P at 25 degrees as at 4 degrees, but did not inhibit the reverse reaction. Therefore, we conclude that E1P was the predominant phosphorylated form of the eel enzyme under the above conditions, and that E1P was converted to E2P and then dephosphorylated by K+.
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology; D004593:Electrophorus; D066298:In Vitro Techniques; D008566:Membranes / Q000201:enzymology; D009840:Oligomycins / Q000494:pharmacology; D010766:Phosphorylation; D012754:Sharks; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D003902:Detergents; D009840:Oligomycins; D000244:Adenosine Diphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase