On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids

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 muM ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (k(app) congruent to 400 s(-1)). v(o) of phosphorylation of CaE1 increased with [ ATP] along a Michaelis-Menten curve (K-m of 15 muM) and was phospholipid-independent. Without Ca2+ preincubation (E-1 + E-2), 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 E-2 --> E-1 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 muM 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 E-2 to E-1, further stimulating the ATPase activity.