Cooperative Association of Actin Protomers and Crosslinked Actin Oligomers in Filaments at Low Ionic Strength.
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概要
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F-Actin treated with a half molar equivalent of the heterobifunctional cross-linker, 4- bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE), produced a population of actin oligomers containing 2-14 or more protomers and a significant amount of uncrosslink-ed actin protomers. The crosslinking reaction is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an adjacent protomer on the genetic helix of F-actin, in a similar manner to N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers were found to form filaments that sediment after high-speed centrifugation, even in a buffer of low ionic strength [G-buffer: 2mM tris-hydroxymethylaminomethane (pH 8. 0), 0.2mM CaCl2, 0.2mM ATP, 0.3mM NaN3, 5mM 2-mercaptoethanol] where affinity between actin protomers usually becomes weak, leading to the depolymerization of native F-actin. By performing the crosslinking reaction in the presence of an environment-sensi-tive fluorescent label, 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which com-petes with BNBA-SE for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were obtained which were also stable in G-buffer. Fluorometric analysis of actin labeled with acrylodan (prodan-actin) in these depolymerization-resistant filaments suggested that the molecular environment around Cys 374 of actin protomers is similar to that of actin protomers in native F-actin in F-buffer (G-buffer plus 0.1M KCl and 1mM MgCl2). When G- actin labeled with acrylodan was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled actin was incorporated into filaments that were resistant to depolymerization in G-buffer. The emission spectrum of the prodan-actin in these filaments measured in G- buffer was almost identical to that of prodan-actin in native F-actin in F-buffer. Our interpretation of this result is that actin protomers are locked into an F-actin-like conformation in the depolymerization-resistant filament by the subunit interactions they make with crosslinked oligomers. We also present evidence which suggests that the depolymerization rate in G-buffer of filaments made with crosslinked oligomers is much slower than that of native actin because the ends of the depolymeriza-tion-resistant filaments are capped with crosslinked oligomers.
- 社団法人 日本生化学会の論文
著者
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Miyata Hidetake
Department Of Physics Faculty Of Science And Technology Keio University
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Kinosita Kazuhiko
Department Of Physics Faculty Of Science And Engineering Waseda University
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MARRIOTT Gerard
Department of Physics, Faculty of Science and Technology, Keio University
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KINOSITA Kazuhiko
Department of Physics, Faculty of Science and Technology, Keio University
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