Abstract
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.
Highlights
Using direct and indirect measurements of divalent cations, we showed that the slow change in fluorescence of 1,5-I-AEDANS-actindirectly paralleled and essentially monitored the exchange of divalent cation on actin (4).Based on this, and therecently recognized fact that theaffinity of actin for divalent cation is much higher than had previously been thought (1, 4, 6-8), we have proposed that divalent cation exchange at thehigh affinity site of actin is best described by
We reported on the derivation of this expression,2and it has been reported without derivation by Nowak et al (1).Experimental data for exchange of divalent cation at intermediate ratios of [Ca]/[Mg] should obey this theoretical relationship if the simple competitive model for divalent cation exchange on actin is correct
If [Mg] = 0 prior to time t = 0, [CaA], = [AT]. This analysis indicates that, independent of the initial conditions, upon adjustment of [Ca] and [Mg] a t timet = 0, divalent cation exchange will proceed at a rate governed by the constant kaPq(Equation 71, which in turn depends upon the specific associatlon and dissociation rate constants for Ca2+and Mg2+ and the ratio [Ca]/ [Mg]
Summary
The major parameter controlling this relationship in the simple competitive exchange model, the ratio of the association rate constants for Ca2+andMg2+ to actin (&dkMg), is found to have a value of about 90. The model appears to be gaining acceptance, some investigators insist on attributing the “Mg-induced slow fluorescence change” in l$-I-AEDANS-actin to divalent cation exchange at a site of moderate affinity for M e (10). The competitive model for divalent cation binding to actin is simple, there areseveral features andexperimental complications which tendto obscure the simplicity. Both k-c, and k-Mg are markedly affected by pH (4)and solution salt conditions (5), as might be expected. Some investigators have suggested that protein-chelator interactions may confound divalent cation exchange measurements (10, 12)
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