The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase.

The effect of the hydrolysis product Pi and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na+ + K+)-activated ATPase was investigated. The hypothesis that (Mg2+ + p-nitrophenyl-P)-supported ouabain binding might be due to Pi release and thus (Mg2+ + Pi)-supported could not be confirmed. The enzyme . ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme . ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or Pi. The presence or absence of Na+ during binding has a special influence upon the character of the enzyme . ouabian complex. Without Na+ and in the presence of Tris ions the complex obtained with (Mg2+ + Pi) and that obtained with (Mg2+ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na+ concentration diminished the level of Pi-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme . ouabain complexes, however, now resembled the form obtained with (Na+ + ATP), as judged from their dissociation rates and the K+ sensitivity of their decay. The complexes obtained at a high Na+ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K+ to the resuspension medium. The most stable enzyme . ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate of Pi facilitated complex formation. The presence of K+ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na+ concentration. It is proposed that the different ouabain dissociation rates reflect different reactive states of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.