Behavior of the Ca2+ transport sites linked with the phosphorylation reaction of ATPase purified from the sarcoplasmic reticulum.
Both the formation and decay of phosphorylated ATPase, and Ca2+ release from and rebinding to transport sites alpha sites) on the enzyme have been investigated in the pre- and post-steady states by means of rapid mixing acid quench technique and a stopped flow technique, respectively. At a relatively high concentration of enzyme (10 muM protein) and a low concentration of free Ca2+ (1.42 muM), rapid changes in the Ca2+ affinity of the transport sites could be monitored with the use of a Ca2+ indicator dye, Arsenazo III. As the enzyme becomes phosphorylated, Ca2+ is released. In the early stages, the ratio of Ca2+ released to acid-stable phosphoenzyme is less than 2, while at the maximum of Ca2+ release, which occurs later than the maximum phosphorylation, it is greater than 2. Assuming that phosphorylation of the enzyme releases both Ca2+ bound to it (Ikemoto, N. (1975) J. Biol. Chem. 250, 7219), these data are consistent with the sequential formation of two acid-stable intermediates differing in Ca2+ affinity and a third acid-labile phosphorylated species of low Ca2+ affinity. The changes in Ca2+ affinity are probably related to changes in the structure of the ATPase transport protein that are involved in the Ca2+ translocation in the intact sarcoplasmic reticulum. As the ATP is being used up, Ca2+ rebinding occurs concomitantly with the decay of phosphoenzyme. The comparison of the time courses of Ca2+ rebinding with that of dephosphorylation in the post-steady state suggests that interactions among phosphorylated and nonphosphorylated ATPase molecular may also be involved in the Ca2+ affinity changes.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010750:Phosphoproteins; D000251:Adenosine Triphosphatases; D002118:Calcium