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 Vmax without affecting apparent affinities of the ATP sites. In the presence of 20 microm ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (kapp congruent with 400 s-1). vo of phosphorylation of CaE1 increased with [ATP] along a Michaelis-Menten curve (Km of 15 microm) and was phospholipid-independent. Without Ca2+ preincubation (E1 + E2), vo 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. kapp for dephosphorylation was 57 s-1 at 0 microM ATP; that rate was further increased by ATP. Steady-state [EP] x kapp 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.
Filomatori
Claudia V
CV
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Junín 956, 1113 Buenos Aires, Argentina.
Rega
Alcides F
AF
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013552:Swine
D010743:Phospholipids; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases