Structure of the 265-Kilodalton Complex Formed upon EDC Cross-Linking of Subfragment 1 to F-Actin

A. L. Andreeva, O. A. Andreev, J. Borejdo

Research output: Contribution to journalArticle

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Abstract

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 actc-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 actc-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 actc-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175–185-kDa actc-S1 complex (Skactin ratio = 1). The ability of the myosin head to bind to one or to two actins suggests that during an active stroke the myosin head may first bind to one and then to two monomers in F-actin, producing a ∼10-nm shift between thick and thin filaments.

Original languageEnglish
Pages (from-to)13956-13960
Number of pages5
JournalBiochemistry
Volume32
Issue number50
DOIs
StatePublished - 1 Jan 1993

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Actins
Myosins
Digestion
Carbodiimides
Subtilisin
Peptides
Actin Cytoskeleton
Molecular mass
Adenosine Triphosphatases
Muscle
Stroke
Monomers
Muscles
actin subfragments
Molecules

Cite this

Andreeva, A. L. ; Andreev, O. A. ; Borejdo, J. / Structure of the 265-Kilodalton Complex Formed upon EDC Cross-Linking of Subfragment 1 to F-Actin. In: Biochemistry. 1993 ; Vol. 32, No. 50. pp. 13956-13960.
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Structure of the 265-Kilodalton Complex Formed upon EDC Cross-Linking of Subfragment 1 to F-Actin. / Andreeva, A. L.; Andreev, O. A.; Borejdo, J.

In: Biochemistry, Vol. 32, No. 50, 01.01.1993, p. 13956-13960.

Research output: Contribution to journalArticle

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T1 - Structure of the 265-Kilodalton Complex Formed upon EDC Cross-Linking of Subfragment 1 to F-Actin

AU - Andreeva, A. L.

AU - Andreev, O. A.

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N2 - 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 actc-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 actc-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 actc-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175–185-kDa actc-S1 complex (Skactin ratio = 1). The ability of the myosin head to bind to one or to two actins suggests that during an active stroke the myosin head may first bind to one and then to two monomers in F-actin, producing a ∼10-nm shift between thick and thin filaments.

AB - 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 actc-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 actc-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 actc-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175–185-kDa actc-S1 complex (Skactin ratio = 1). The ability of the myosin head to bind to one or to two actins suggests that during an active stroke the myosin head may first bind to one and then to two monomers in F-actin, producing a ∼10-nm shift between thick and thin filaments.

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