title: The role of Mg2+ and Ca2+ in the simultaneous binding of vanadate and ATP at the phosphorylation site of sarcoplasmic reticulum Ca2+-ATPase.
abstract: The sarcoplasmic reticulum Ca2+-ATPase was reacted with vanadate in the presence of Mg2+ and EGTA, and the effect of Ca2+, Mg2+ and ATP on the kinetics of vanadate release from the enzyme vanadate complex was studied after dilution with vanadate-free media. Ca2+ increased, whereas ATP decreased the rate of vanadate release. In absence of free Mg2+ in the release media ATP was bound to the vanadate-reacted Ca2+-ATPase with high affinity (Kd 4-5 microM), and full saturation with ATP resulted in complete inhibition of vanadate release. In media containing free Mg2+, where ATP predominantly was present as MgATP, binding of the nucleotide to vanadate-reacted Ca2+-ATPase occurred with low apparent affinity. Mg2+ alone did not affect the rate of vanadate release. At saturating ATP concentrations the release rate in the presence of free Mg2+ was less inhibited than in its absence. These results indicate that uncomplexed ATP interacts with the same Mg2+ at the catalytic site, which is involved in formation of the enzyme-vanadate complex (EMgV), and thereby hinders dissociation of vanadate. Destabilization of the complex by free Mg2+ may be caused by the presence of an additional magnesium ion in the catalytic site together with ATP.
authors.forename: J P
authors.initials: JP
authors.lastname: Andersen
authors.forename: J V
authors.initials: JV
authors.lastname: Møller
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism*
chemical_list: D014639:Vanadium; D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium