The influence of membrane charge on the kinetic properties of Na,K-ATPase.
Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.5 for Na+ was small, (K0.5Na,TPP = 0.180 mM and K0.5Na,TPB = 0.07 mM), indicating only a minor influence of membrane charge on Na+ binding to site I. Site II: Compared to control conditions, more Na+ was required to inhibit ATP-hydrolysis and to increase the steady-state level of ADP-sensitive phosphoenzyme when TPP+ was present, and the opposite was observed with TPB-, indicating a strong influence of membrane charge on the Na+ occupancy of site II. Site III: TPP+ induced a significant decrease both in the rate of K-dependent dephosphorylation of preformed E32P and in the K+ affinity. The effect of TPP+ on the ATP hydrolysis rate strongly resembled the effect of decreasing [KCl]. The results indicated a pronounced effect of adding positive charge to the lipid environment of site III.(ABSTRACT TRUNCATED AT 250 WORDS)
Klodos
I
I
Institute of Biophysics, University of Aarhus, Denmark.
Plesner
L
L
D000244:Adenosine Diphosphate / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D009861:Onium Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013775:Tetraphenylborate / Q000494:pharmacology
D009861:Onium Compounds; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; D013775:Tetraphenylborate; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C013289:tetraphenylphosphonium