Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase.
Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca(2+)-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25 degrees C. The time course of phosphorylation with ATP of the enzyme associated with Ca(2+), probably the true phosphorylation reaction, showed a fast phase (k(app) near 400 s(-1)) followed by a slow phase (k(app)=23 s(-1)). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4 ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (k(app)=93 s(-1)) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (k(app)=30 s(-1)) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (k(app)=220 s(-1)) and slow (k(app)=20 s(-1)) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.
Bredeston
Luis M
LM
Instituto de Química y Fisicoquímica Biológicas (Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas), Facultad de Farmacia y Bioquímica, Junín 956, 1113 Buenos Aires, Argentina.
Rega
Alcides F
AF
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents / Q000737:chemistry; D007700:Kinetics; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000737:chemistry
D002147:Calmodulin; D003902:Detergents; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010743:Phospholipids; D011092:Polyethylene Glycols; C024990:asolectin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium