ATP binding by the isolated N-P domains of the plasma membrane Ca2+ pump

MONTES M. GUADALUPE; MAZZITELLI LUCIANA ROMINA; ADAMO HUGO PEDRO

San Javier, Tucuman

Encuentro; XLI Reunion Anual de la Sociedad Argentina de Biofisica; 2012

Sociedad Argentina de Biofisica

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.