The effect of ATP and Ca2+ on the phosphatase activity of the purified plasma membrane Ca2+ pump.

RINALDI DEBORA ELENA; MAZZITELLI LUCIANA ROMINA; ADAMO HUGO PEDRO

Rosario

Congreso; XXXIX Annual Meeting of Argentinean Biophysical Society SAB 2010, workshop CeBEM, structural biology in Latin America, 3rd Latin American Protein Society Meeting; 2010

XXXIX Annual Meeting of Argentinean Biophysical Society SAB 2010, workshop CeBEM, structural biology in Latin America, 3rd Latin American Protein Society Meeting

Like other ion motive P-type ATPases the Ca2+ transporter from plasma membranes (PMCA) can hydrolyze non nucleotidic phosphate esters like p-nitrophenyl phosphate.  This phosphatase activity has been associated with the E2-like conformations of the enzyme which are those devoid of Ca2+.   In agreement with this idea we have recently shown that the pNPPase activity of the purified PMCA from pig erythrocytes is maximal in the absence of Ca2+, and that Ca2+   inhibits the pNPPase by stabilizing the E1 form of the enzyme1.  ATP is also an inhibitor of the pNPPase activity probably by competing with pNPP at the hydrolysis site2.  However previous studies have shown that ATP can rescue the enzyme from Ca2+ inhibition by forcing the enzyme to cycle and to reach a new steady state of E2-like conformations.  We have measured the pNPPase activity at a high concentration of ATP (3 mM) as a function of the Ca2+ concentration. We found that when reconstituted in phosphatidylcholine the pNPPase activity in the absence of Ca2+ was very low and it increased with increasing concentrations of Ca2+. However  if the enzyme was  reconstituted with acidic lipids, the pNPPase activity in the absence of Ca2+ was higher and it remained constant up to 10 mM Ca2+. This behavior suggests that when the enzyme is activated by acidic lipids   the deprivation of E2 by the binding of Ca2+ is nearly compensated by the formation of E2 trough cycling so that the relative amount of E2-like forms remains constant.