Ouabain Reaction with Microsomal (Sodium‐plus‐Potassium)‐Activated Adenosinetriphosphatase

Microsomal (Na + + K + )‐activated adenosinetriphosphatase from eel electroplax binds ouabain in a slow reaction. The rate of binding is modified by enzyme ligands, including nucleotides, orthosphosphate, p ‐nitrophenylphosphate, carbamylphosphate, Mg 2+ , Na + , and K + . The actions of these ligands on the enzyme were studied by measuring their effects on the initial rate of ouabain binding. The stimulatory actions ATP, ADP, and P i depend on the formation of enzyme · Mg · phospholigand complexes. Free nucleotides and P i are inhibitory. Two alternative pathways for ouabain binding are confirmed: (a) Na + ‐stimulated, ATP‐dependent, and (b) Na + ‐inhibited, P i ‐dependent. In addition, it is shown that the Na + ‐stimulated path may depend on any of the organic phosphate ligands, including p ‐nitrophenylphosphate, carbamylphosphate and ADP. In contrast, the Na + ‐inbibited path is supported only by MgHPO 4 or free Mg 2+ . There is evidence for non‐identical Na + ‐binding sites: the [Na] 0.5 value for stimulation is 20 mM while that for inhibition is 4 mM. In the presence of both ATP and P 1 , the Na+‐potentiated‐ATP path is favored. K + inhibits both pathways reversibly and exhibits competition with Na + and with MgHPO4. ATP decreases the enzyme affinity for K + while Na + plus ATP increase the affinity for K + . Since the P i · enzyme complex is observed only in the absence of both Na + and K + , it is considered a side path not related to the catalytic cycle. The influences of the ligands on ouabain binding may be related to their effects on the enzyme catalytic cycle. There are two general hypotheses for the ligand effects on ouabain reactivity of (Na + + K + )‐ATPase. One, proposed by Sen et al. in 1969, is based on the formation of phosphorylenzyme. The observations are also consistent with an alternative hypothesis which is proposed as follows: (a) the enzyme reactivity with ouabain is increased by the formation of an enzyme · Mg · phospholigand complex regardless of whether the enzyme is also phosphorylated, (b) Na + ‐activated enzyme has increased affinity for the organic phosphates, which may be considered as substrates, and decreased affinity for P i and free Mg 2+ , and (c) K + ‐activated enzyme has decreased affinity for all the ligands.