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

Abstract

The occlusion of Rb+ by Na, K-ATPase was 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. 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 Na+ stabilized the enzyme in the E1 form. The extra radioactivity carried through the columns when nucleotides and Na were absent was taken as a measure of the amount of Rb occluded within the enzyme (in the E2 form) when it emerged from the resin. 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 .fwdarw. E1) that allows the occluded Rb+ to escape. At 20.degree. C, and in the absence of nucleotides, it is .apprx. 0.1 s-1. The rate constant for Rb release was the same in a Na-containing as in a K-containing medium. The opposite effects of Na+ and K+ 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. The rate constant for Rb release was greatly increased by ATP and by ADP. Both nucleotides appeared to act at low-affinity sites and without phosphorylating the enzyme. Orthovanadate, in the presence of Mg2+, stabilized the enzyme in the occluded-Rb (E2Rb) form. Ouabain, in the presence of Mg2+, prevented the occlusion of Rb+. The amount of Rb occluded by the enzyme was measured as a function of Rb concentration. At saturating Rb concentrations .apprx. 3 Rb+ can be occluded per phosphorylation site (or per ouabain-binding site). The occluded-Rb form of the enzyme can also be formed by allowing Rb+ to catalyze the hydrolysis of phosphoenzyme generated by the addition of ATP to enzyme suspended in a high-Na medium. The properties of the occluded-Rb form of the enzyme, and of the 2 routes that can lead to its formation, suggest that an analagous occluded-K form plays a central role in the transport of K+ through the Na-K pump. This hypothesis is supported by a detailed consideration of the probable magnitudes of the rate constants of the individual reactions making up the 2 routes.