Evidence for the ordered release of rubidium ions occluded within the Na,K-ATPase of mammalian kidney.

When Na,K-ATPase containing occluded rubidium ions is exposed to orthophosphate, in the presence of magnesium ions, there is a rapid release of half or all of the occluded ions. This behaviour is observed irrespective of whether the occluded-rubidium form of the enzyme is generated by putting the unphosphorylated enzyme in a sodium-free medium containing rubidium ions, or by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme made by adding ATP to enzyme suspended in a medium containing sodium and magnesium ions. The release of occluded rubidium ions by orthophosphate requires the presence of magnesium, presumably because phosphorylation is necessary. Whether the addition of orthophosphate causes the rapid release of all or of half of the occluded rubidium depends on whether free rubidium (or potassium, thallium or (probably) caesium ions) are present in the medium at the time the orthophosphate is added. In the absence of free ions of these species, all of the occluded rubidium is released. In their presence (in adequate concentration), only half of the occluded rubidium is released. The relative effectiveness of the different potassium congeners in preventing the rapid release of 50% of the occluded rubidium when orthophosphate is added is: thallium greater than rubidium greater than potassium greater than caesium. Lithium and sodium are ineffective even at high concentrations, and sodium ions strongly antagonize the effect of free rubidium ions. In a sodium-free, Tris medium, the concentration of free rubidium ions necessary for a half-maximal effect is about 30 microM. In a medium containing 250 microM-free rubidium, the concentration of sodium necessary to reduce the effect of free rubidium by 50% is about 500 microM. These figures are compatible with the hypothesis that the free rubidium or other ions act at the potassium-loading sites at the extracellular face of the pump. By starting with enzyme occluding unlabelled rubidium, and using 86Rb-labelled free rubidium, it is possible to show that the free ions that prevent the rapid release of half of the occluded ions themselves become occluded. These experiments are significant in two ways. First, they provide direct evidence for the existence of a second route for the release of occluded rubidium (and therefore presumably of occluded potassium) ions. Secondly, they seem to require that the release of occluded ions by this route occurs in an ordered fashion.