Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Abstract

SUMMARY1. The occlusion of rubidium ions by Na, K-ATPase has been investigated by suspending enzyme prepared from pig kidney outer medulla in media containing low concentrations of 86Rb, forcing the suspensions rapidly through small columns of cation-exchange resin, and measuring the amounts of radioactivity emerging from the columns.2. When the suspension media contained 2 mM-ATP or ADP, or 15 mm-NaCl, the amounts of radioactivity emerging from the columns were greatly (and similarly) reduced, presumably because both nucleotides and sodium ions stabilized the enzyme in the E1 form. (See p. 19 for definition of E1 and E2). The extra radioactivity carried through the columns when nucleotides and sodium were absent was taken as a measure ofthe amount of rubidium occluded within the enzyme (in the E2 form) when it emerged from the resin.3. By varying the flow rate, and therefore the time spent by the enzyme on the resin, and relating this to the amount of radioactivity emerging from the columns, we have been able to estimate the rate constant for the conformational change (E2 -+ E1) that allows the occluded rubidium ions to escape. At 20 'C, and in the absence of nucleotides, it is about 0-1 s-1.4. The rate constant for rubidium release was the same in a sodium-containing as in a potassium-containing medium. The opposite effects of sodium and potassium ions on the poise of the equilibrium between the E1 and the E2 forms of the enzyme must, therefore, be due solely to opposite effects of these ions on the rate of conversion of E1 to E2.5. The rate constant for rubidium release was greatly increased by ATP and by ADP. Both nucleotides appeared to act at low-affinity sites and without phosphorylating the enzyme.6. Orthovanadate, in the presence of magnesium ions, stabilized the enzyme in the occluded-rubidium (E2Rb) form.7. Ouabain, in the presence of magnesium ions, prevented the occlusion of rubidium ions.8. We have measured the amount of rubidium occluded by the enzyme as a function of rubidium concentration, and estimate that at saturating rubidium concentrations about three rubidium ions can be occluded per phosphorylation site (or per ouabain-binding site).I. M. GL YNN AND D. E. RICHARDS 9. We have found that the occluded-rubidium form of the enzyme can also be formed by allowing rubidium ions to catalyze the hydrolysis of phosphoenzyme generated by the addition of ATP to enzyme suspended in a high-sodium medium.10. The properties of the occluded-rubidium form of the enzyme, and of the two routes that can lead to its formation, suggest that an analogous occluded-potassium form plays a central role in the transport of potassium ions through the sodiumpotassium pump. This hypothesis is supported by a detailed consideration of the probable magnitudes of the rate constants of the individual reactions making up the two routes.

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

“…This value is in contrast with the proposed stoichiometry of 1 Ca 2ϩ transported per ATP hydrolyzed by the PMCA (20,21,22). In our hands 4 (not shown, but see Ref. 26), the preparation of erythrocyte ghosts, which is not highly enriched in PMCA, presents serious problems for filtration through Millipore-type filters, and washing out of Ca 2ϩ from the ghosts using the ionophore A23187 is slow and inefficient.…”

The molecular size required varies according to the reaction step round the sodium pump cycle

“…Instead, when the assay is performed at physiological ATP levels, something the size of cy2, or perhaps cud, is the active species. The implication is that for ATP to bind at a lowaffinity site to catalyse the release of the K+-occluded form [27] (fig.4, path a-b-d-f), another a-chain is required, but not if the release occurs spontaneously at a low rate (at catalytic ATP concentrations, path a-b-d-e). A target size of 117 kDa has been reported [ 151 for an ATPindependent Rb+-Rb+ exchange catalysed by the Na+ pump, which seemingly shares steps d-e of the same path.…”

“…dephosphoenzyme [26,27] (step f). Since only one ATP-binding site has been identified so far in the cr-chain and none in the &chain [lo], it was decided to determine whether low-affinity ATP effects required the presence of a second a-chain.…”

The energy coupled exchange of Na+ for K+ across the cell membrane

“…Addition of Na' leads to a transition to ErNaJ, with a Ko.5 for Na+ of a few mM. Addition of K + to EZ leads to a con~orrnatiol~ from which Ki has a very low rate of release, an occluded form, Ez'(K2) [75]. The affinity of EZ for K' is low, but as the eq~iilibriul~ between ElK2 and Ez'(&) is poised towards Ez'(Kz), the apparent affinity of Ez for K' is high with a Ko.5 in the FM range.…”