High affinity divalent cation exchange on actin. Association rate measurements support the simple competitive model.
Each actin molecule has one high affinity site which binds a divalent cation. It has been proposed that an isomerization of the actin molecule is involved in divalent cation exchange at this site ("isomerization model," Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886); we have maintained that exchange is by a simple competitive mechanism (Estes, J. E., Selden, L. A., and Gershman, L. C. (1987) J. Biol. Chem. 262, 4952-4957). Here, using fluorescent-labeled actin, we measure the apparent rate constant for exchange (kapp) as a function of the ratio of free Ca2+ and Mg2+ concentrations, ([Ca]/[Mg]), and show that both models are consistent with the data. The major parameter controlling this relationship in the simple competitive exchange model, the ratio of the association rate constants for Ca2+ and Mg2+ to actin (kCa/kMg), is found to have a value of about 90. We have verified this parameter by direct measurements of kCa and kMg, finding that kCa = 1.9 x 10(7) M-1 s-1 and kMg = 2.3 x 10(5) M-1 s-1, consistent with the characteristics of the Ca2+ and Mg2+ aquo ions. The corresponding parameter derived from the isomerization model is not verifiable. We conclude that high affinity divalent cation exchange on actin proceeds by a simple competitive mechanism.
Gershman
L C
LC
Medical Service, Veterans Administration Medical Center, Albany, New York 12208.
Selden
L A
LA
Estes
J E
JE
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D008274:Magnesium / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D009132:Muscles / Q000378:metabolism; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D008274:Magnesium; D002118:Calcium