Interaction between G-actin and myosin subfragment-1 probed by covalent cross-linking.
The topography of rapid equilibrium complexes formed between G-actin and myosin subfragment-1, which are the first kinetic intermediates in the polymerization process into F-acto-S1 filaments, has been probed by chemical cross-linking. In the absence of ATP, cross-linking of G-actin-S1 complexes by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) yielded a major 165-170-kDa and a fainter 200-205-kDa doublet polypeptide. The actin:S1 molar ratio was 1 in the EDC-cross-linked complexes, using either double labeling techniques or the method combining EDC + N-hydroxysuccinimide. Chemical cleavages of the covalently cross-linked complexes by formic acid and N-hydroxylamine (Sutoh, K. (1983) Biochemistry 22, 1579-1585) showed that in the main cross-linked 165-kDa polypeptide, the 1-12 acidic N-terminal region of actin was covalently linked to the lysine-rich region connecting the central 50-kDa domain to the C-terminal 20-kDa domain of S1, as in F-acto-S1 complexes. G-actin, but not F-actin, was covalently cross-linked to S1 by N,N'-paraphenylenedimaleimide (p-PDM). A major 195-kDa and a minor 165-kDa polypeptide were obtained, could be separated from actin and S1 by DEAE-cellulose chromatography, and did not exhibit actin-activated Mg-ATPase activity. Both EDC-cross-linked and p-PDM-cross-linked complexes between G-actin and S1 could be incorporated into F-acto-S1 decorated filaments. The C-terminal cysteine 374 of actin is involved in the p-PDM cross-linked 195-kDa complex. Accordingly, a covalent photocross-linked 200-kDa conjugate was formed between S1 heavy chain and benzophenone-G-actin, obtained by covalent modification of Cys374 by benzophenonemaleimide (Tao, T., Lamkin, M., and Scheiner, C. J. (1985) Arch. Biochem. Biophys. 240, 627-634). These results demonstrate that (i) G-actin-S1 and F-actin-S1 complexes display a large similarity in the EDC-cross-linked electrostatic close contacts and (ii) a change in the environment of Cys374 is linked to the polymerization into F-actin-S1 decorated filaments.
Combeau
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Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Didry
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Carlier
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D000199:Actins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D005033:Ethylmaleimide; D005022:Ethyldimethylaminopropyl Carbodiimide