EFFECTS OF THE TYPE OF DIVALENT-CATION, CA2-AFFINITY SITE AND OF THE IONIC COMPOSITION OF THE SOLUTION ON THE STRUCTURE OF F-ACTIN( OR MG2+, BOUND AT THE HIGH)

F-actins containing either Ca2+ or Mg2+ at the single high-affinity si te for a divalent cation differ in their dynamic properties [Carlier ( 1991) J. Biol. Chem. 266, 1-4]. In an attempt to obtain information on the structural basis of this difference, we probed the conformation o f specific sites in the subunits of Mg- and Ca-F-actin with limited pr oteolysis by subtilisin and trypsin. The influence of the kind of poly merizing salt was also investigated. At high proteinase concentrations required for digestion of actin in the polymer form, subtilisin gives a complex fragmentation pattern. In addition to the earlier known cle avage between Met(47) and Gly(48) in the DNAse-I-binding loop, cleavag e of F-actin between Ser(234) and Ser(235) in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) F EES Lett. 365, 149-151]. Here we show that actually a larger segment, comprising residues 227-235, is removed and the bond between Leu(67) a nd Lys(68) in subdomain 2 is split in both G- and F-actin, and that th e differences in the fragmentation patterns of the G- and F-forms are accounted for by the protection of the bond 47-48 in F-actin. The subt ilisin and trypsin cleavage sites in segment 61-69, subtilisin sites i n segment 237-235 and trypsin sites between Lys373 and Cys(374) were l ess accessible in Mg-F-actin than in Ca-F-actin. These are intramolecu lar effects, as similar changes were observed on Ca2+/Mg2+ replacement in G-actin. The cation-dependent effects, in particular those on segm ent 61-69, were however less pronounced in F-actin than in G-actin. Th e results suggest that substitution of Mg2+ for Ca2+, and KCl-induced polymerization of CaATP-G-actin, bring about a similar change in the c onformation of subdomain 2 of the monomer. The presence of Mg2+ at the high-affinity site also resulted in an increased protection of the bo nd 47-48. This latter appears to be an intermolecular effect because i t is specific for F-actin. The susceptibility to subtilisin and trypsi n was also strongly influenced by the kind and concentration of polyme rizing salt. The digestion patterns suggest that the exposure and/or f lexibility of the regions containing the cleavage sites diminish with enhancement of the ionic strength of the solution. The results are dis cussed in terms of the current models of F-actin.