EFFECT OF REPLACEMENT OF THE TIGHTLY BOUND CA2+ BY BA2+ ON ACTIN POLYMERIZATION

G-actin has a single tight-binding (high-affinity) site for divalent c ations per mole of protein, whose occupancy is important for the stabi lity of the molecule. Different tightly bound divalent cations differe ntly influence the polymerization properties of actin. The tightly bou nd metal ion easily exchanges for free exogenous cations, Moreover, bi ochemical and structural evidence demonstrates that actin, in both the G- and F-forms, assumes different conformations depending on the meta l ion bound with high affinity in the cleft between two main domains o f the molecule, In this work, we used proteolytic susceptibility to de tect possible local conformational alterations of the actin molecule f ollowing a brief incubation of Ca-G-actin with barium chloride and eth ylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We found that substitution of Ba2+ for the tightly bound Ca2+ affects th e regions around Arg-62 and Lys-68 in subdomain 2 of G-actin, as judge d from inhibition of tryptic cleavage at these residues. Using the flu orescent chelator Quin-2, we observed that about 0.95 mol of Ba2+ is r eleased per 1 mol of actin. We also examined the effect of replacement of the tightly bound Ca2+ by Ba2+ on actin polymerization. With respe ct to Ca-actin, Ba-actin shows an increased polymerization rate, mainl y due to its enhanced nucleation and a higher critical concentration. (C) 1998 Academic Press.