The Journal of General Physiology, Vol 104, 33-55, Copyright © 1994 by The Rockefeller University Press
ADP+orthophosphate (P(i)) stimulates an Na/K pump-mediated coefflux of P(i) and Na in human red blood cell ghosts
R Marin and JF Hoffman
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
The Na/K pump in human red blood cells that normally exchanges 3 Nai for 2
Ko is known to continue to transport Na in a ouabain-sensitive and
ATP-dependent manner when the medium is made free of both Nao and Ko.
Although this Na efflux is called "uncoupled" because of removal of ions to
exchange with, the efflux has been shown to be comprised of a coefflux with
cellular anions. The work described in this paper presents a new mode of
operation of uncoupled Na efflux. This new mode not only depends upon the
combined presence of ADP and intracellular orthophosphate (P(i))i but the
Na efflux that is stimulated to occur is coeffluxed with (P(i))i. These
studies were carried out with DIDS- treated resealed red cell ghosts,
suspended in buffered (NMG)2SO4, that were made to contain, in addition to
other constituents, varying concentrations of ADP and P(i) together with
Na2 SO4, MgSO4 and hexokinase. While neither ADP nor P(i) was effective
alone, ouabain- sensitive uncoupled Na efflux, (measured with 22Na) could
be activated by [ADP+P(i)] where the K0.5 for ADP in the presence of 10
mmol (P(i))i/liter ghosts was 100-200 mumol/liter ghosts and the K0.5 for
(P(i))i, in the presence of 500 mumol ADP/liter ghosts was 3-4 mmol/liter
ghosts. [ADP+P(i)] activation of this Na efflux could be inhibited by as
little as 2 mumol ATP/liter ghosts but the inhibition could be relieved by
the addition of 50 mM glucose, given entrapped hexokinase. While
ouabain-sensitive Na efflux was found to be coeffluxed with P(i) (measured
with entrapped [32P]H3PO4), this was not so for SO4 (measured with 35SO4).
The stoichiometry of Na to P(i) efflux was found to be approximately 2 to
1. Na efflux as well as (P(i))i efflux were both inhibited by 10 mM Nao
(K0.5 approximately equal to 4 mM). But, whereas 20 mM Ko (K0.5
approximately equal to 6 mM) inhibited the efflux of (P(i))i, as would be
expected from previous work, Na efflux was actually increased. When Ko
influx was measured in this situation there was a 1 for 1 exchange of Nai
for Ko, that is, of course, downhill with respect to the gradient of each
ion. Surprisingly AsO4 was unable to replace P(i) for activation of Na
efflux but Na efflux could be inhibited by vanadate and oligomycin. In
terms of mechanism, it is likely that ADP acts to promote the formation of
the phosphoenzyme (EP) by (P(i))i that would otherwise be inhibited by
Nai.(ABSTRACT TRUNCATED AT 400 WORDS)
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