INVESTIGADORES
MONTES Monica Raquel
congresos y reuniones científicas
Título:
Rb+ deocclusion from the vanadate-bound Na,K-ATPase is not parallel to the E2-E1 transition
Autor/es:
SPIAGGI ALEJANDRO, MONTI J.L, ROSSI RC AND MONTES MR.
Lugar:
California
Reunión:
Congreso; 13th International P-type ATPase meeting, Na,K-ATPase and Related P-ATPases: Structure, Biology and Medicine,; 2011
Resumen:
Rb+ deocclusion from the vanadate-bound Na,K-ATPase
is not parallel to the E2-E1 transition.
Alejandro J.
Spiaggi, José L. Monti, Rolando C. Rossi, and Mónica R. Montes
Facultad de
Farmacia y Bioquímica, UBA. Junín 956 (1113) Buenos Aires, Argentina.
E-mail:
mmontes@qb.ffyb.uba.ar
Vanadate is a well-known
inhibitor of the activity of P-type ATPases (1) that is currently being used in
structural studies to mimic the bipyramidal transition state of the E2P form (2). The present study explores
the effect of vanadate on the transition between the E2 and E1 conformations
and its relation with the intermediate with Rb+ occluded by the
direct route in the Na,K-ATPase. Conformational transitions were measured
through eosin fluorescence changes and occluded Rb+ was quantified
by a rapid filtration technique (3), under presteady-state and equilibrium
conditions.
Control experiments
performed in the absence of vanadate showed that the time course of the E2®E1
conformational change promoted by Na+ is parallel to the release of
Rb+ from the occluded state. The addition of vanadate (0.1 mM) in the presence of
Mg2+ (1.75 mM)
cancels the transition from E2 to E1 induced by Na+ whereas Rb+
deocclusion still occurs at a significant rate. Mg2+‑vanadate
increased the K0.5 of Rb+ for the occluded state from 5
to 40 mM reaching the same maximal level. Our
results suggest that even when Mg2+‑vanadate stabilizes an E2-like state, this is not a guarantee
of a more stable Rb+-occluded form.
This work was supported by grants
from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional
de Investigaciones Científicas y Técnicas and Universidad de Buenos Aires,
Argentina.
1- Cantley L.C. Cantley L.G.
and Josephson L., J.Biol.Chem. (1978)
253:7361-7368.
2- Clausen J.D., McIntosh D.,
Woolley D. and Andersen J.P., J.Biol.Chem.
(2011) 286: 11792-11802
3- Rossi R.C., Kaufman S.B.,
González-Lebrero R.M., Nørby J.G. and Garrahan P.J., Anal. Biochem. (1999) 270:276-285.