IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Detection of occluded Rb+ intermediates during ATP hydrolysis catalyzed by Na+/K+-ATPase in nominally Na+-free media
Autor/es:
J. L. E. MONTI; P. G. SCHVARTZ; P. J. GARRAHAN; R. C. ROSSI
Lugar:
Rosario, Argentina
Reunión:
Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
The membrane protein Na+/K+-ATPase
couples the hydrolysis of 1 ATP molecule to the exchange of 3 cytoplasmic Na+
for 2 extracellular K+. Mg2+ is an essential activator of
the catalysis. The catalytic mechanism remains unclear. It is known that during
the catalytic cycle the enzyme becomes phosphorylated and that the transported
cations become occluded (trapped within the enzyme). K+-occluded
intermediates of the Na+/K+-ATPase can be obtained either
by the direct route, without formation of phosphoenzyme, or by the physiological
route, after K+-stimulated dephosphorylation of the
phosphoenzyme. Several cations can replace K+, including Rb+
(1-3). Previous studies (4) show that as [Na+] is increased at a
fixed [Rb+], occluded Rb+ and ouabain-sensitive ATPase
activity increase along a sigmoidal curve with an initial slope equal to zero,
as expected if 3 Na+ were needed for ATP phosphorylation of the
pump. However, a small but significant ATPase activity is measured when no Na+
is added to the reaction media. Moreover, under these conditions, there is an
increase and then a decrease of ATPase activity as [Rb+] increases. The
inhibition of ATPase activity at high [Rb+] is explained by the inhibition
of enzyme phosphorylation due to formation of occluded Rb+
intermediates through the direct route. On the other hand, the increase in
ATPase activity observed at low [Rb+] can be explained by assuming
that Rb+ enhances dephosphorylation of the same phosphointermediate
that is formed through Na+‑activated phosphorylation. Because such
dephosphorylation should lead to Rb+ occlusion, we have measured steady-state
levels of occluded Rb+ at 25 °C using a purified
preparation of pig kidney Na+/K+-ATPase in nominally free
Na+ media (with less than 10 mM Na+) containing
2.5mM ATP, imidazole-HCl 25mM, pH=7.4, and different concentrations of [86Rb+]RbCl
and CholineCl (an inert salt added in order to keep constant the ionic strength
at 170mM), with 0.5mM free Mg2+ or without Mg2+. A small
but significant increase of occluded Rb+ in the presence of Mg2+
is observed, suggesting that Na+ activated phosphorylation leads to the
same Rb+-sensitive phosphointermediate that is formed in the assayed
conditions. Since only H+ and Li+ have been reported as
possible Na+ analogs (5,6), it remains an open question how enzyme
is phosphorylated.
Supported with
grants from: UBACYT, ANPCYT, and CONICET.
References
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[2] Forbush B, J. Biol. Chem., 1987, 262,
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[3] Rossi RC, González-Lebrero RM, Kauffman SB,
Garrahan PJ, J. Gen. Physiol., 2005, 126, 29a-30a.
[4] Monti JLE, Schvartz PG, González-Lebrero RM,
Kauffman SB, Rossi RC,Garrahan PJ, J.
Gen. Physiol., 2005, 126,
28a-29a.
[5] Beaugé L, Biochim,
Biophys. Acta, 1978, 527, 472 - 484.
[6] Hara Y, Nakao M, J. Biol. Chem., 1986,
261, 12655 - 12658.