Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes.

摘要: To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of and temperature, purified (Na,K)-ATPase from dog kidney eel electroplax, monitored protein conformation, possible subunit interactions, fluidity membrane with fluorescent probes. The p-nitrophenylphosphatase activities were inhibited reversibly pressures below 1.5 kilobars (eel enzyme) 2.5 (dog enzyme). Above these pressures, enzymes inactivated irreversibly. plots 1n(activity) versus curvilinear; this indicates that reversible inhibition involves two or more rate-limiting steps. calculated activation volumes varied temperature larger for activity compared to activity. fluorescence polarization three hydrophobic probes decreased increasing (10-36 degrees C) increased (10(-3)-1.5 kilobars), reversibly, without any evidence lipid phase transition. Plots showed an inverse relationship; was directly related Pressure had no effect on cardiac glycoside nor efficiency resonance energy transfer between either donor acceptor glycosides specifically bound ouabain sites different alpha-subunits, tryptophan probe. These results suggest dissociation dimeric alpha-subunits is not pressure, glycoside-complexed conformation stabilized pressure. It concluded decreases hinders conformational transitions associated steps reaction. proposed ion-bound -occluded forms are because they occupy smaller volume.