PHOSPHATE FROM THE PHOSPHOINTERMEDIATE (EP) OF THE HUMAN RED-BLOOD-CELL NA K PUMP IS COEFFLUXED WITH NA, IN THE ABSENCE OF EXTERNAL-K/

This study is concerned with Na/K pump-mediated phosphate efflux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump tha t occurs in the absence of external Na-0 and K-0. Because this efflux (measured with Na-22) is also inhibited by 5 mM Na-0, the efflux can b e separated into a Na-0-sensitive and a Na-0-insensitive component. Pr evious work established that the Na-0-sensitive efflux is actually com prised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as (SO4)-S-35). The present work focuses on the Na-0-insensiti ve component in which the principal finding is that orthophosphate (P- i) is coeffluxed with Na in a ouabain-sensitive manner. This P-i efflu x can be seen to occur, in the absence of K-0, in both DIDS-treated in tact cells and resealed red cell ghosts. This efflux of P-i was shown to be derived directly from the pump's substrate, ATP, by the use of r esealed ghosts made to contain both ATP and P-i in which either the AT P or the P-i were labeled with, respectively, [gamma-P-32]ATP or [P-32 ]H3PO4. (These resealed ghosts also contained Na, Mg, P-i, SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspend ed usually in (NMG)(2)SO4 at pH 7.4.) It was found that P-32 was only coeffluxed with Na when the P-32 was contained in [gamma-P-32]ATP and not in [P-32]H3PO4. This result implies that the P-32 that is released comes from ATP via the pump's phosphointermediate (EP) without commin gling with the cellular pool of P-i. K-0 (as K2SO4) inhibits this P-32 efflux as well as the Na-0-sensitive (SO4)-S-35 efflux, with a K-0.5 of 0.3-0.4 mM. The K-0.5 for inhibition of P-i efflux by K-0 is not in fluenced by Na-0, nor can Na-0 act as a congenor for K-0 in any of the flux reactions involving K-0. The stoichiometry of Na to SO4 and Na t o P-i efflux is similar to 2:1 under circumstances where the stoichiom etry of Na effluxed to ATP utilized is 3:1. From these and other resul ts reported, it is suggested that there are two types of uncoupled Na efflux that differ from each other on the basis of their sensitivity t o Na-0, the source (cellular vs substrate) and kind of anion (SO4 vs P -i) transported. Whether more than one kind of alpha (or beta) isoform is involved to account for the pump's ability to transport P-i is not known.