Passive rubidium fluxes mediated by Na‐K‐ATPase reconstituted into phospholipid vesicles when ATP‐ and phosphate‐free

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Abstract
Phospholipid vesicles reconstituted with Na-K-ATPase from pig kidney, show slow passive pump-mediated 86Rb fluxes in the complete absence of ATP and phosphate. The Rb fluxes are inhibited in vesicles prepared from enzyme pre-treated with either ouabain or vanadate ions. Rb fluxes through Na-K pumps oriented inside-out or right-side out by comparison with the normal cellular orientation can be distinguished by effects of vanadate on one or both sides of the vesicle. 86Rb uptake into Rb-loaded vesicles represents a 86Rb-Rb exchange. The maximal rate of exchange through inside-out and right-side out oriented pumps is equal, suggesting a random arrangement of the pumps across the vesicle membrane. This Rb-Rb exchange is half-saturated on inside-out and right-side out pumps at about 0.6 and 0.2 mM-external Rb, respectively. 86Rb uptake into Rb-free vesicles represents a net Rb flux. The Rb uptake through inside-out pumps has a maximal rate about equal to the Rb-Rb exchange, half-saturates at an external Rb concentration of roughly 0.5 mM, and shows evidence for co-operativity. Net Rb uptake through right-side out pumps is very slow, and half-saturates at roughly 0.1 mM external Rb. K ions at low concentrations in the exterior medium stimulate 86Rb uptake, but at high concentrations, inhibit. Na ions in the exterior medium always inhibit 86Rb uptake. K ions are probably transported in co-operative fashion together with Rb ions, while Na ions block the Rb fluxes. The presence of Rb congeners at the vesicle interior raises the 86Rb uptake through inside-out pumps with the decreasing order of effectiveness: Li > Na > Cs > K > Rb. Stimulation by Na ions involves a Rb-Na exchange. Turnover numbers were estimated from parallel measurement of Na/K pump mediated fluxes and amount of covalent phosphoenzyme. In units of mol of ion/mol of phosphoenzyme per s at 20.degree. C the following values were obtained: ATP-dependent Na-Rb exchange, 43; (ATP + phosphate)-stimulated Rb-Rb exchange, 7. For (ATP + phosphate)-independent fluxes: Rb-Rb exchange 0.25; net Rb uptake 0.15 and Rb-Na exchange 0.65. Mg ions in the exterior medium inhibited both net and exchange Rb fluxes through inside-out pumps in a manner antagonistic with respect to Rb. Mg and vanadate ions inhibit the Rb fluxes in a synergistic fashion. The results are interpreted in terms of a model in which net and exchange 86Rb fluxes occur via conformational transitions between form E1 which binds Rb at the cytoplasmic face of the protein, the form E2 (Rb)occ containing occluded Rb ions and a form E2 which binds Rb at the extracellular face of the protein. A kinetic analysis identifies rate-limiting steps of the transport cycle by making use of transport data in combination with values of rate-constants for conformational transitions observed directly in isolated Na-K-ATPase.

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