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J. Biol. Chem., Vol. 278, Issue 25, 22265-22271, June 20, 2003

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On the Mechanism of Activation of the Plasma Membrane Ca²⁺-ATPase by ATP and Acidic Phospholipids^*

Claudia V. Filomatori  and Alcides F. Rega 

From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Junín 956, 1113 Buenos Aires, Argentina

The activation of purified and phospholipid-depleted plasma membrane Ca²⁺-ATPase by phospholipids and ATP was studied. Enzyme activity increased with [ATP] along biphasic curves representing the sum of two Michaelis-Menten equations. Acidic phospholipids (phosphatidylinositol (PI) and phosphatidylserine (PS)) increased V_max without affecting apparent affinities of the ATP sites. In the presence of 20 µM ATP, phosphorylation of the enzyme preincubated with Ca²⁺ (CaE₁) was very fast (k_app 400 s^–1). v_o of phosphorylation of CaE₁ increased with [ATP] along a Michaelis-Menten curve (K_m of 15 µM) and was phospholipid-independent. Without Ca²⁺ preincubation (E₁ + E₂), v_o of phosphorylation was also phospholipid-independent, but was slower and increased with [ATP] along biphasic curves. The high affinity component reflected rapid phosphorylation of CaE₁, the low affinity component the E₂ E₁ shift, which accelerated to a rate higher than that of the ATPase activity when ATP was bound to the regulatory site. Dephosphorylation of EP did not occur without ATP. Dephosphorylation increased along a biphasic curve with increasing [ATP], showing that ATP accelerated dephosphorylation independently of phospholipid. PI, but not phosphatidylethanolamine (PE), accelerated dephosphorylation even in the absence of ATP. k_app for dephosphorylation was 57 s^–1 at 0 µ_M ATP; that rate was further increased by ATP. Steady-state [EP] x k_app for dephosphorylation varied with [ATP], and matched the Ca²⁺-ATPase activity measured under the same conditions. Apparently, the catalytic cycle is rate-limited by dephosphorylation. Acidic phospholipids stimulate Ca²⁺-ATPase activity by accelerating dephosphorylation, while ATP accelerates both dephosphorylation and the conformational change from E₂ to E₁, further stimulating the ATPase activity.

Received for publication, March 14, 2003 , and in revised form, March 24, 2003.

^* This work was supported by grants from the Consejo Nacional de Invetigaciones Científicas y Técnicas, Argentina, the Universidad de Buenos Aires, and the Ministerio de Salud, Argentina. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a fellowship from the Universidad de Buenos Aires.

To whom correspondence should be addressed. Tel.: 5411-49648289 (ext. 123); Fax: 5411-49625457; E-mail: rega{at}qb.ffyb.uba.ar.

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