Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase is a pivotal residue for cation-induced conformational changes.
The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.
Johnson
C L
CL
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, OH 45267-0575, USA.
Kuntzweiler
T A
TA
Lingrel
J B
JB
Johnson
C G
CG
Wallick
E T
ET
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002414:Cations, Monovalent / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D010042:Ouabain / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D012756:Sheep; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D002414:Cations, Monovalent; D007527:Isoenzymes; D018698:Glutamic Acid; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium