Abstract
Polymerization of G-actin in the presence of salt and phalloidin was blocked by treatment of G-actin with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N, N' -p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). The m- and d-actins retained the ability to bind to myosin heads with apparent dissociation constants of 3-8×10-6 and 3-5×10-7M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. However, m-actin formed a 2:1 m-actin-head complex, suggesting the presence of at least two latent actin-binding sites on a myosin head. ATP weakens only 2-to 6-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins to myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBS-treated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin binding sites on a myosin head: one is responsible for binding of F-, m-, and d-actins, the second for binding of F- and m-actins, and the third for binding of F-actin at least in the presence of ATP. F-Actin binding to the third site may in some way block the first and second binding sites.
