The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum.
The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP  with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003956:Dialysis; D008274:Magnesium; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium