Properties of the recombinant human plasma membrane Ca2+ pump purified from yeast.

CURA CAROLINA; ADAMO HUGO PEDRO

Montevideo, Uruguay

Conferencia; 6 International Conference of Biological Physics; 2007

      8- Transporters, receptors and channels   Properties of the recombinant human plasma membrane Ca2+ pump purified from yeast.   Carolina Cura 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: carocura@hotmail.com    The Ca2+ extrusion by the Ca2+ transporter from plasma membrane (PMCA) is essential for maintaining the intracellular Ca2+ homeostasis. The PMCA is a scarce protein comprising about 0.5 % of the protein of the membrane and an efficient heterologous expression system is needed to advance its structure-function studies (Adamo et al., 1992).   We have used the yeast Saccharomyces cerevisiae to express functional PMCA in the range of 25 ug of purified recombinant protein (yrPMCA) per liter of culture (Bredeston and Adamo, 2004). We have conducted experiments aimed to characterize the recombinant PMCA obtained from yeast by comparison with the original protein from human erythrocytes (ePMCA). Both proteins were purified using an identical protocol using a calmodulin affinity column after solubilization of the membranes with  the detergent C12E10 in the presence of 20% glycerol as stabilizer.  At 37 °C the yrPMCA supplemented with phosphatidylcholine (PC) was able to hydrolyze ATP at a rate of 0.3 mmol/mg/min in the absence of Ca2+ and the addition of 1 mM Ca2+ had a negligible effect on the rate of ATP hydrolysis which was in this condition 0.4 mmol/mg/min. In contrast the ePMCA hydrolyzed ATP at a rate of 0.1 mmol/mg/min but the addition of Ca2+ raised the activity to 3 mmol/mg/min. When PC was replaced by a mixture of acidic lipids (BE), the Ca2+-ATPase activity of ePMCA increased about 2 fold while that of yrPMCA was more than 10 times higher. These results show that under optimal conditions the solubilized yrPMCA has about half of the ATPase activity of the native ePMCA and its activity is particularly sensitive to the composition of the lipidic environment. Adamo, H.P, Verma, A.K, Sanders, M.A, Heim, R., Salisbury, J.L., Wieben, E.D., Penniston, J.T. Biochem. J. 1992, 285:791-797  Bredeston, L.M. and Adamo, H.P. J. Biol. Chem 2004 , 279:41619-41625     Supported by grants from ANPCyT, CONICET and UBA.