title: Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model.
abstract: Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.
mesh_terms: D000199:Actins / Q000378:metabolism*; D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008433:Mathematics; D008958:Models, Molecular; D009282:Naphthalenesulfonates; D011817:Rabbits
chemical_list: D000199:Actins; D000634:Aminoquinolines; D002413:Cations, Divalent; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D008274:Magnesium; C035739:Quin2; D002118:Calcium