The interaction of amines with the occluded state of the Na,K-pump.
We have studied the effect of various amines on the rate of release of 86Rb from the occluded state of dog kidney Na,K-ATPase formed by pre-incubation of the enzyme with 86Rb. In the presence of MgPi, various amines act like K+ or Rb+ in blocking the release of 86Rb from one of two sites for occlusion (the "s" site). Of 38 amines tested, tetrapropylamine and various benzyl amines exhibit the highest affinity; the K1/2 for these compounds is 2-5 mM. In the presence of ATP, when 86Rb is presumably released towards the intracellular face of the pump in the normal mode of operation, 86Rb release is blocked by the presence of amine, but only if the amine is also included in a preincubation with MgPi. The data are consistent with a model in which the interaction of amine with one of the transport sites (the "f" site) prevents the E2----E1 transformation that is stimulated by ATP. When 86Rb deocclusion from the f site has occurred in the presence of amine, the lone 86Rb at the s site can be released in the presence of ATP if the amine is removed from the medium. This suggests that a single 86Rb ion at the s site can be released to the intracellular face of the membrane, and therefore that transport can occur with only one K+ site occupied. The amine that blocks release of one 86Rb ion does not itself become occluded: (a) The interaction of amine and ATP is only seen when both ligands are present in the medium; (b) the effects of amines are not "remembered" after a brief exposure to a rinse medium; (c) with the vanadate-inhibited enzyme, benzyltriethylamine and tetrapropylamine are only weakly effective in blocking 86Rb release from the s site; and (d) organic cations exhibit very low affinity in competition with 86Rb for occlusion at equilibrium. Thus the results are consistent with the idea that monofunctional amines block by binding to the f site but that, unlike K+ and Rb+, they do not become occluded. In contrast, at equilibrium ethylenediamine prevents 86Rb occlusion in a competitive manner, suggesting the possibility of occlusion of the bifunctional amine.
Forbush
B
B
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000588:Amines / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002794:Choline / Q000494:pharmacology; D004285:Dogs; D005029:Ethylenediamines / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D017616:Magnesium Compounds; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D000588:Amines; D005029:Ethylenediamines; D017616:Magnesium Compounds; D010710:Phosphates; D000644:Quaternary Ammonium Compounds; C052377:tetrapropylammonium; C030781:magnesium phosphate; C031234:ethylenediamine; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D012413:Rubidium; D002794:Choline