INFLUENCE OF INTRAMEMBRANE ELECTRIC CHARGE ON NA,K-ATPASE

Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphe nylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were s tudied with membrane-bound enzyme from bovine brain, pig kidney, and s hark rectal gland. Na+ and K+ interactions with the inward-facing bind ing sites, monitored by eosin fluorescence and phosphorylation, were n ot influenced by lipophilic ions. Phosphoenzyme interactions with extr acellular cations were evaluated through K+-, ADP-, and Na+-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-in sensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the r ate coefficient for dephosphorylation of the K-sensitive phosphoenzyme , 3) the Na+ affinity and the rate coefficient for Na+-dependent depho sphorylation, Pre steady state phosphorylation experiments indicate th at the subsequent occlusion of extracellular cations was prevented by TPP+, TPB- had opposite effects. Effects of lipophilic ions on the tra nsition between phosphoenzymes were significantly diminished when Nawas replaced by N methyl-D-glucamine or Tris(+), but were unaffected b y the replacement of Cl- by other anions. Lipophilic ions affected Na- ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accord ance with their effects on the partial reactions. Effects of lipophili c ions appear to be due to their charge indicating that Na+ and K+ acc ess to their extracellular binding sites is modified by the intramembr ane electric field.