IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Effects of ADP on the Na+/K+-ATPase
Autor/es:
P. G. SCHVARTZ; J. L. E. MONTI; 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 Na+/K+-ATPase is an
intrinsic membrane protein which couples the transport of Na+ and K+
with the hydrolysis of ATP. The pump can
hydrolyze ATP in the absence or in the presence of K+ through the so-called
Na+-ATPase and Na+/K+-ATPase cycles,
respectively. These can be described in terms of the Post-Albers model, which
postulates that, in presence of K+ or Rb+ (a K+-like
cation), the Rb+ occluded intermediate can bind ATP to a regulatory,
activating site [1].
In a previous work [2], we have shown that the time
courses of the reaction intermediates could only be described considering a
competitive effect of ADP for the ATP binding sites in E1 and in E2. To test this hypothesis, we investigated the
effects of ADP on the steady-state level of the Na+/K+-ATPase
reaction intermediates during Na+-ATPase and Na+/K+-ATPase
activities.
We first determined the concentration of E2P,
measuring the fluorescence change of the RH421 dye [3] at 25 °C in a medium containing 150 mM NaCl and 0.5 mM free Mg2,
and 25 mM
Imidazole-HCl, pH 7.4, at different concentrations of ATP and ADP. Results show
that, during Na+-ATPase activity, ADP could act as a mixed-type
inhibitor, competing with ATP in E1 and acting as a product
inhibitor in the reverse reaction E1P+ADP->E1ATP.
Additionally, we measured the concentration of Rb+
occluded through the standard quench flow technique [4] or by the fluorescence
change of the RH421, in the same medium as that described above, plus 6 mM RbCl. Results show that ADP, besides the effects observed
in absence of Rb+, binds to the E2 form of the pump
acting in lieu of ATP as a partial activator of the ATPase activity.
The ability of ADP to increase the deocclusion rate
was confirmed measuring the initial velocity of deocclusion at different [ADP],
the results showing a hyperbolic relationship with a K0.5 for ADP of
750 mM.
The results were adequately fitted by the equations
derived from the Post-Albers mechanism and the values of the rate constants were
estimated. We observed that the rate constant for the product inhibition caused
by ADP is small enough as to be neglected at the micromolar concentrations of
ATP used in the experiments described previously [2].
[1] Glynn, I. M., and Karlish, S. J., 1990, Annu Rev Biochem, 59, 171-205.
[2] Schvartz
P.G., Monti J.L.E.., González-Lebrero R.M., Kaufman S.B., Garrahan P.J. and Rossi
R.C., 2005, J Gen
Physiol, 126, 30a-31a.
[3] Schvartz P.G., Monti J. L. E., González-Lebrero R. M.,
Kaufman S. B., Garrahan P. J., and Rossi R. C., 2004, XXXIII Reunion de la SAB, Mar del Plata
[4] Rossi, R.C.,
Kaufman, S.B., González-Lebrero, R.M., Norby, J. G., and Garrahan P.J., 1999, Anal.
Biochem., 270, 276285.
[Supported by CONICET, Agencia Nacional de Promoción
Científica y Tecnológica and Universidad de Buenos Aires]