The E2 in equilibrium E1 transition of the Ca2(+)-ATPase from plasma membranes studied by phosphorylation.
The relative abundance of the two conformers (E1 and E2) of the Ca2(+)-ATPase of plasma membranes and the rates of their interconversion were estimated measuring the initial velocity of phosphorylation of the Ca2(+)-ATPase in pig red cell membranes at 37 degrees C. This was based on the hypothesis that only E1 catalyzes phosphorylation from ATP. In the absence of ligands near 90% of the Ca2(+)-ATPase was in the E2 conformation. Ca2+ shifted the equilibrium toward E1. The K0.5 of Ca2+ for this effect was 15 microM, suggesting that it acted at the transport site. The conversion of E2 into E1 was slow (t1/2 = 911 s) while the conversion of E1 into E2 was faster (t1/2 less than or equal to 60 s). Mg2+ accelerated the E2----E1 reaction lowering its t1/2 to 0.25 s. In the presence of 4 mM Ca2+ t1/2 was 7.8 s, as if at this concentration to some extent Ca2+ replaced Mg2+ in accelerating the E2----E1 reaction. Results suggest that in intact membranes at 37 degrees C Ca2+ stabilized E1 and that the Ca2(+)-induced E2----E1 transition was strongly accelerated by Mg2+. Both cations were effective at near physiological concentrations and in the absence of other ligands like ATP or calmodulin that could also modify the reaction. After partial proteolysis with trypsin the Ca2(+)-ATPase behaved during phosphorylation as if it were E1.
Adamo
H P
HP
Instituto de Química y Fisicoquímica Biológicas (Universidad de Buenos Aires-Consejo Nacional de Investigaciones, Científicas y Técnicas), Facultad de Farmacia y Bioquímica, Argentina.
Rega
A F
AF
Garrahan
P J
PJ
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D008954:Models, Biological; D010766:Phosphorylation; D013552:Swine
D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium