Structure of the 265-kilodalton complex formed upon EDC cross-linking of subfragment 1 to F-actin.
The conventional model of force generation in muscle requires the presence of at least two different contact areas between the myosin head (S1) and the actin filament. It has been found that S1 has two sites available for carbodiimide cross-linking, but it is generally believed that the myosin head can be cross-linked to only one actin through either site. We provide here, for the first time, evidence that one S1 can be cross-linked to two separate actin molecules. The covalent complex of one S1 with two actins was found to have an apparent molecular mass of 265 kDa. The formation of the 265-kDa acto-S1 complex was strongly dependent on the ratio of S1 to actin. Limited tryptic digestion converted the 265-kDa product into the 240-kDa complex by releasing a 27-kDa N-terminal S1 fragment. Limited subtilisin digestion of the 265-kDa covalent acto-S1 complex yielded 29-, 93-, and 66-kDa peptides which corresponded to the 29-kDa N-terminal domain of S1, actin-44-kDa (central domain of S1) and actin-22-kDa (C-terminal domain of S1) complexes, respectively. These peptides could be generated only if a single S1 has been cross-linked to two separate actins. The 265-kDa acto-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175-185-kDa acto-S1 complex (S1:actin ratio = 1).(ABSTRACT TRUNCATED AT 250 WORDS)
Andreeva
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AL
Baylor Research Institute, Baylor University Medical Center, Dallas, Texas 75226.
Andreev
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OA
Borejdo
J
J
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D014357:Trypsin
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D005022:Ethyldimethylaminopropyl Carbodiimide