The effects of the solvents deuterated water (2H2O) and dimethyl sulfoxide (Me2SO) on [3H]ouabain binding to (Na+,K+)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg2++ATP+Na+). In contrast, both solvents stimulated type II (i.e., Mg2++Pi-, or Mn2+-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg2++Na++ATP, 75% in the Mg2++Pi system, and in the presence of Mn2+ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, 2H2O inhibition of type I ouabain binding was dependent on Na+ concentration in the reaction and was reduced as Na+ was elevated. Contact of the enzyme with Me2SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na++ATP first and then to Me2SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me2SO and 2H2O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H2O, and E1 enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H2O at the active enzyme center and E2 enzyme conformation. This postulation of a role of H2O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.
Bibliographical noteFunding Information:
This work is supported by funds from Medical Research Fund of the U.S. Veterans Administra-
- (Rat brain)
- Deuterated water
- Ouabain binding
- Solvent effect