PROPERTIES OF THE RECOMBINANT PLASMA MEMBRANE Ca2+ PUMP PURIFIED FROM YEAST 

CURA CAROLINA; ADAMO HUGO PEDRO

Rosario, Argentina

Congreso; XXXV Reunion Anual de la Sociedad Argentina de Biofisica (SAB); 2006

Sociedad Argentina de Biofisica (SAB)

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. After the initial cloning work that lead to the isolation of a full-length cDNA coding the human PMCA isoform 4xb (Adamo y col. 1992) various heterologous over expression systems have been tried in order obtain amounts of PMCA protein suitable for functional and structural studies. We have recently showed that the PMCA can be expressed in a functional form in the yeast Saccharomyces cerevisiae (Bredeston and Adamo, 2004). Here we report experiments aimed to characterize the recombinant PMCA obtained from yeast (yrPMCA) by comparison with the original protein from human erythrocytes (ePMCA). Both proteins were purified using an identical protocol. Membranes isolated from yeasts or human erythrocytes were solubilized with 0.68 g C12E10 per g of total membrane protein in a media containing 1 mM PMSF, 2 mM DTT, 0.5 mM CaCl2, 20 mM MOPS, 130 mM KCl, 1 mM MgCl2, 20% glycerol and the PMCA from the solubilizate was purified using a calmodulin affinity column. 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 highlight the existence of functional differences between the native and the recombinant enzyme purified from yeast cells. Under optimal conditions the solubilized yrPMCA had about half of the ATPase activity of the native ePMCA and its activity was 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.