Na+ movement in a single turnover of the Na pump.

Abstract

Ouabain-sensitive 22Na efflux from right-side-out membrane vesicles prepared from dog kidney has been examined with a time resolution of 30 msec. The vesicles are preloaded with 22Na and caged ATP [P3-1-(2-nitro)phenylethyl adenosine triphosphate], so that transport by the Na pump can be initiated by light. After a brief illumination, which releases less ATP than the number of catalytic sites, a burst of 22Na extrusion is observed corresponding to a single turnover of the Na pump. By the use of a rapid filtration apparatus, with which a continuous record of the rate of efflux is obtained, it has been possible to resolve the efflux burst in the time range of 20-1500 msec. The rate of efflux rises rapidly, but not instantaneously, to a peak and then decays, with a time constant of approximately equal to 6 sec-1 at 15 degrees C. The time course of Na efflux is unaffected by extracellular K+, as predicted by models of the Na pump in which Na is released early in the cycle. Unphotolyzed caged ATP is found to bind to the catalytic site of Na,K-ATPase, in competition with ATP that is produced in the flash, and the possibility has not been excluded that dissociation of unphotolyzed caged ATP and binding of ATP are involved in the Na efflux time course. It seems most likely that binding of ATP and translocation of 22Na are involved in the increase in the 22Na efflux rate in the single turnover and that the release of transported 22Na from extracellular pump sites limits the slow decay.