External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts.

Abstract

Coupled active transport of Na+ and K+ across cellular plasma membranes is mediated by (Na+ + K+)-stimulated Mg2+-dependent ATPase. Active cation transport by this Na pump involves a cyclic Na-dependent phosphorylation of the enzyme by intracellular ATP and hydrolytic dephosphorylation of the phosphoenzyme, stimulated by K+ (ref. 1). In human red blood cells, skeletal muscle and squid axons, replacement of extracellular K by Na results in a ouabain-sensitive efflux of Na coupled to an influx of extracellular Na. There is apparently no net Na movement nor net hydrolysis of ATP. The rate of Na:Na exchange is stimulated by increased levels of ADP and exchange transport is not observed in cells totally depleted of intracellular ATP. These characteristics suggest that the biochemical mechanism underlying the Na exchange mode of the Na pump involves phosphorylation of the enzyme by ATP (which requires intracellular Na) followed by its dephosphorylation by ADP. Such a reaction has been observed in partially purified (Na+ + K+) ATPase from a variety of sources and its dependence on Na concentration has been described (although not previously for the red cell enzyme). In the present work, intracellular ATP:ADP exchange reaction was initiated by photoreleased ATP following brief irradiation at 350 nm of ghosts containing caged-ATP. The ouabain-sensitive component of the ensuing ATP:ADP exchange reaction shows a biphasic response to extracellular Na. External Na in the range 0--10 mM has an inhibitory effect whilst increasing concentrations beyond this range stimulate the rate of exchange in a roughly linear fashion up to 100 mM Na. These results represent the first direct demonstration of the sidedness of the effects of Na on this partial sequence in the overall enzyme cycle and bear a qualitative resemblance to the Na effects on the Na-ATPase which occur in the absence of intracellular ADP in human red blood cells.