Abstract
Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K(+)-, ADP-, and Na(+)-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na(+)-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB- had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl- by other anions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field.
Highlights
Lipophilic ions affected Na-ATPase, Na,K·ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions
Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field. (Andersen et at., 1978; Flewelling and Hubbell, 1986a, 1986b)
In the present work we applied biochemical techniques supported by measurements of conformational changes by eosin fluorescence to describe which partial reactions in the Na,KATPase reaction cycle are affected by lipophilic ions
Summary
Lipophilic ions affected Na-ATPase, Na,K·ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. The ions are located a few angstroms below the membrane surface (Andersen et at., 1978), and they modifY the electrostatic potential profile inside the membrane dielectric (Andersen et at" 1978; Flewelling and Hubbell, 1986a; 1986b). It has been shown by Buhler et at. The authors suggested that the external cation binding sites are formed as deep wells, i.e. K+ binds to and Na + is released from "sites that are located inside the membrane dielectric" (Liiuger, 1991a). This article must be hereby marked "advertisement" in accordance with 18
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