Evidence for the existence of two ATP-sensitive Rb+ occlusion pockets within the transmembrane domains of Na+/K+-ATPase.
A trypsin-digested Na+/K+-ATPase that has lost ATPase activity and about half of its protein content retains an essentially intact beta-subunit, the 10 transmembrane domains of the alpha-subunit, and the full capacity to occlude Na+ and Rb+ (a congener of K+). When this preparation was incubated at 37 degrees C in the absence of Rb+, it lost half of its Rb+ occluding capacity and two-thirds of its Na+ occluding capacity. Comparison of the Rb+ occlusion-deocclusion kinetics of the digested enzyme before and after partial inactivation indicated that (a) the affinities of the labile and the stable halves of occluded Rb+ were the same; (b) occlusion and deocclusion rates of the stable pool were lower than those of the labile pool; (c) ATP at a low affinity site (K0.5 = 25-300 microM) increased deocclusion rate in the stable pool and occlusion rate in the labile pool; (d) Na+ increased Rb+ deocclusion rate of the sum of the two pools but not that of the stable pool; and (e) occlusion and deocclusion rates of both pools were decreased by ouabain. These findings suggest that (a) the peptide complex of the digested enzyme contains two distinct but interacting cation occlusion pockets, one occluding two Na+ or one Rb+, and the other occluding one Na+ or one Rb+; (b) this peptide complex that is devoid of the catalytic ATP site retains an allosteric ATP site; and (c) the access channels of the two pockets are regulated differently by ATP but similarly by ouabain. Analyses of the gel electrophoretic patterns of the digested enzyme and the N termini of the appropriate bands showed that inactivation of the labile occlusion pocket was accompanied by 60-70% loss of two alpha-fragments containing H3-H4 and H5-H6 transmembrane domains. This and the previously established interactions among the transmembrane helices of alpha- and beta-subunits suggest that one occlusion pocket is associated with H3-H6 domains and that the other is located within a complex of beta-subunit and two alpha-fragments containing H1-H2 and H7-H10 transmembrane domains.
Liu
L
L
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008, USA.
Askari
A
A
D000818:Animals; D004285:Dogs; D012413:Rubidium; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium