A myosin head can interact with two chemically modified G-actin monomers at ATP-modulated multiple sites.
It has been reported that chemically modified [with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric form and retains the ability to bind (and make chemical cross-links) to myosin head [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032; Arata, T. (1991) J. Biochem. (Tokyo) 109, 335-340]. Here, the interaction between MBS-G-actin and myosin subfragment 1 (S1) has been further studied by proteolytic susceptibility and chemical cross-linking. Two moles of MBS-actin monomers bound to 1 mol of myosin heads or S1 with different affinities. The first binding of MBS-G-actin to S1 strongly protected a 27-kDa/50-kDa junction of S1 heavy chain from trypsin digestion and also weakly protected a 50-kDa/20-kDa junction. The second binding protected a 50-kDa/20-kDa junction more strongly. ATP weakened these bindings more than 10-fold. MBS-G-actin was cross-linked to S1 by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, producing the 175-185-kDa doublet bands similar to those of F-actin and S1. The first binding produced a complex migrating at 175 kDa on gels [Hozumi, T. (1992) Biochemistry 31, 10071-10073] and the second binding further produced an 185-kDa complex, suggesting that two binding sites correspond to two spatially separated cross-linking sites. MBS-G-actin was also cross-linked by MBS to S1 when the first actin binds, producing only 180-kDa complex. In the presence of ATP or ADP, an 140-kDa complex was produced together with the 180-kDa complex, suggesting shifting of the binding site.
Arata
T
T
Department of Biology, Graduate School of Science, Osaka University, Japan. arata@bio.sci.osaka.u.ac.jp
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; C066969:maleimidobenzoyl-G-actin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate