Functional significance of the binding of one myosin head to two actin monomers.
The functional significance of the interaction of one myosin head (S1) with two actin monomers was investigated by comparing the properties of the cross-linked monomeric and filamentous actin-S1 complexes. S1 was cross-linked to monomeric actin (G-actin) either in the absence or in the presence of DNase I by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binary G-actin-S1 and ternary DNase I-G-actin-S1 complexes were then purified by a combination of ion exchange and gel filtration columns. Both the binary and the ternary complexes were characterized by negligible, though different, Mg(2+)-ATPase activities of 0.018 and 0.006 s-1s(-1), respectively. Using fluorescence, light-scattering, and ultracentrifugation experiments, we found that only the binary G-actin-S1 complex could be polymerized in the presence of 2 mM MgCl2. Electron microscopic analysis of the cross-linked filamentous complex showed fully decorated filaments with the arrowhead pattern characteristic of the non-covalent complex in the rigor state. Such a 100% cross-linked F-actin-S1 complex exhibited a Mg(2+)-ATPase activity of 6.2 +/- 0.8 s-1, slightly lower than the maximum velocity of the non-cross-linked complex of 8.6 +/- 0.8 s-1, but comparable to the 6.9 +/- 0.6 s-1 obtained for a partially (35%) cross-linked complex. These results implied that the activation of S1 ATPase by actin requires the interaction of S1 with a second actin monomer within the thin filament. They also suggested that the full activation of the filamentous complex is not dependent on the degree of saturation of the thin filament by myosin.
Lheureux
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Centre de Recherches de Biochimie Macromoleculaire, CNRS-U9008, INSERM-U249, Montpellier, France.
Chaussepied
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P
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001704:Biopolymers; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D008854:Microscopy, Electron; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D001704:Biopolymers; D003432:Cross-Linking Reagents; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins