Ca2+ - INDEPENDENT HYDROLYTIC ACTIVITIES OF THE Ca2+ PUMP FROM PLASMA MEMBRANE 

MAZZITELLI LUCIANA ROMINA; ADAMO HUGO PEDRO

Rosario, Argentina

Congreso; XXXV Reunion Anual de la Sociedad Argentina de Biofisica; 2006

Sociedad Argentina de Biofisica

8- Transporters, receptors and channels   Ca2+-independent hydrolytic activities of the Ca2+ pump from  plasma membrane   Luciana R. Mazzitelli and Hugo P. Adamo Instituto de Química y Fisicoquímica Biológicas (IQUIFIB) Departmento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina. e-mail: lrmazzitelli@yahoo.com.ar    The Ca2+ pump of plasma membrane (PMCA) is a member of the family of P-type ATPase family of energy transducers that couple the uphill movements of ions with ATP hydrolysis. The currently accepted E1E2 model for the reaction cycle of the PMCA supports the existence of two distinct conformational states,  E1 that can react with micromolar concentrations of ATP to form a high-energy phosphoenzyme E1P,  and E2 that can be phosphorylated by inorganic phosphate to give a low-energy phosphoenzyme E2P. The equilibrium between E1 and E2 is controlled by Ca2+ that binds to E1 with high affinity and therefore displaces the E1E2 equilibrium towards E1. On the basis of this model the ATP hydrolysis by the PMCA depends on the formation of the E1Ca intermediate and hence is Ca2+ dependent. Nowadays the Ca2+ independent ATP hydrolysis is not considered linked to the PMCA and is usually subtracted from the total hydrolytic activity and ignored.  We have recently reported that the ability of the PMCA to hydrolyze p-nitrophenylphosphate is maximal in the absence of Ca2+ (1). Because this activity did not required Ca2+, it  was attributed to the E2 conformation of the enzyme.  After this we have investigated whether the PMCA in the absence of Ca2+ may be also able to catalyze the hydrolysis of  ATP. To this end we measured the ATP hydrolysis in a  standard reaction media containing  3 mM ATP, 4mM MgCl2, 100 mM KCl, 20mM HEPES (pH 7.2),  5 mg of solubilized PMCA purified from pig red cells supplemented with 87 mg/ml of phosphatidylserine and 0.17 mg/ml of C12E10. The reaction was carried out at 37°C. In the presence of 21 mM Ca2+ the ATPase activity was 9.5  mmol/mg/min. On the other hand, when the media contained 2.2 mM EGTA and no CaCl2 was added, ATP was hydrolyzed at a low but significant rate of 0.2 mmol/mg/min. A distinct feature of the PMCA is its regulation by autoinhibition (2). The removal of the C- terminal autoinhibitory domain by deletion mutagenesis or limited proteolysis activates the enzyme. The effect of limited proteolysis on the Ca2+-independent ATPase activity of the purified PMCA supplemented with phosphatidylcholine  was investigated. As the digestion of the PMCA with trypsin progressed the rate of Ca2+ independent ATP hydrolysis increased.  These results point out  a previously unnoticed feature of the PMCA that it may hydrolyze ATP at a low rate in the absence of Ca2+. Moreover, both the Ca2+-independent as well as the Ca2+-dependent PMCA activities seem regulated by the autoinhibitory domain. [1]  Mazzitelli, L. R.  and Adamo, H.P., manuscript in preparation.  [2]  Bredeston, L.M. and Adamo, H.P. J. Biol. Chem 2004 , 279:41619-41625   Acknowledgments:  Supported by grants from ANPCyT, CONICET and UBA.