IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
ATP binding by the isolated N-P domains of the plasma membrane Ca2+ pump
Autor/es:
MONTES M. GUADALUPE; MAZZITELLI LUCIANA ROMINA; ADAMO HUGO PEDRO
Lugar:
San Javier, Tucuman
Reunión:
Encuentro; XLI Reunion Anual de la Sociedad Argentina de Biofisica; 2012
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
ATP
binding by the isolated N-P domains of the plasma membrane Ca2+
pump
RESERVADO SAB
M. Guadalupe Montes, Luciana R.
Mazzitelli, and Hugo P. Adamo
IQUIFIB-Departamento de Química Biológica, Facultad de
Farmacia y Bioquímica, UBA. Junin 956, 1113 Ciudad de Buenos Aires.email: lrmazzitelli@hotmail.com
The plasma membrane Ca2+
pump (PMCA) transports Ca2+ against its electrochemical gradient
from the cytosol to the extracellular space, using the energy provided by the
hydrolysis of ATP. The PMCA belongs to the family of autoinhibited P2B-ATPases.
In humans, there are 4 genes coding for PMCA isoforms. The PMCA are proteins of
about 135 kDa with 10 transmembrane segments. We have overexpressed in E. coli, the central portion of the PMCA
molecule that is exposed to the cytosol between transmembrane segments M4 and
M5. After IMAC purification, the protein fragment exhibited the expected
apparent size of 47 kDa in SDS-PAGE. In a reaction media containing 20 mM Hepes
(pH 7.2 at 37°C), 0.5 mM EGTA, 4 mM Mg2Cl, 100 mM ClK, 12 mM pNPP, 5% glycerol,
5 mM sodium azide, and 40 mg
of the purified N-P domain, no pNPP hydrolysis specifically associated with the
N-P domains was detected. In contrast in a similar media containing 3 mM ATP,
an ATPase activity of 0.18 mmol/mg/min
was observed. This activity was independent of the presence or absence of Ca2+.
In the same conditions plus 1 mM
Ca2+, the full lengh PMCA supplemented with phosphatidylcholine
exhibited an ATPase activity of 0.35 mmol/mg/min.
The incubation of the N-P fragment at 25 °C with 10 mM FITC for 10 min resulted
in the incorporation of the fluorescein label to the peptide. Preincubation at 25 °C with 4 mM ATP for 5
min prior to the FITC treatment produced a much lower level of FITC
incorporation. These results show that the isolated NP domain of the PMCA
retains the ability to bind ATP and may still sustain a residual ATPase
activity.
With granst from ANPCyT , CONICET and UBA.