Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion

Pliszka, Barbara; Rędowicz, Maria Jolanta; Strzelecka‐Gołaszewska, Hanna

doi:10.1016/0014-5793(89)81211-6

Cited by 3 publications

(1 citation statement)

References 10 publications

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“…Actin contains a number of weak binding sites for divalent cations ions with affinities which range from 0.018 mM −1 to 6 mM −1 [61–65]. These binding sites should be at least partly occupied in the presence of 5 mM MgCl 2 or 5 mM CaCl 2 .…”

Section: Discussionmentioning

confidence: 99%

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy

Kany¹,

Wolf²,

Kalbitzer³

2002

FEBS Letters

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Mg-F-actin occurs in two conformational states, I and M, where the N-terminal amino acids are either immobile or highly mobile. In the rigor or ADP complex of rabbit myosin S1 with Mg-F-actin the N-terminal acetyl group of actin stays in its highly mobile state. The same is true for the complexes with the myosin motor domain from Dictyostelium discoideum. This excludes a direct strong interaction of the N-terminal amino acids with myosin in the rigor state as suggested. An interaction of the N-terminus of F-actin with myosin is also not promoted by occupying its low-affinity binding site(s) with divalent ions. The N-terminal high-mobility region may be part of a structural system which has evolved for releasing inadequate stress applied to the actin filaments ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Section: Discussionmentioning

confidence: 99%

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy

Kany¹,

Wolf²,

Kalbitzer³

2002

FEBS Letters

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New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage

Bonet-Kerrache

Harricane²,

Audemard³

et al. 1990

Biochemistry

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The head of the myosin molecule (i.e., subfragment 1 with a heavy chain of 95 kDa) is usually obtained by chymotryptic cleavage in the presence of a divalent cation chelator. In the present work, we used another specific proteolytic enzyme, thrombin, to produce a limited cut within the myosin molecule, resulting in a new species of N-terminal fragment. Treatment of skeletal muscle myosin yielded a 97-kDa split heavy chain associated with intact light chains, corresponding to a single cut. The ATPase activities of this new S-1 derivative were slightly affected by the breakdown. It recognized actin in an ATP-dependent manner, as expected, with an affinity 2-5 times higher than that of the usual chymotryptic S-1 preparation but with a very different electron microscopic pattern. Functional differences are noted, and we involve them more precisely in relation to possible structural aspects of the additional C-terminal segment extending the usual S-1 heavy chain from 95 to 97 kDa.

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Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

Rędowicz

Sobieszek

Strzelecka‐Gołaszewska

1990

European Journal of Biochemistry

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It was previously shown that tryptic digestion of subfragment 1 (Sl) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH,-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25"C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH,-terminus is involved in the communication between the sites of nucleotide and actin binding.We also show here for the first time that the S1-S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain-produced S1 devoid of the regulatory light chain) is also temperaturedependent but insensitive to nucleotides and actin. It is suggested that the temperature-dependent alteration in the flexibility of the head-rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J . Mol. Biol. 182,[271][272][273][274][275][276][277][278][279] Biochem. 177,, may contribute to the temperature dependence of some steps in the cross-bridge cycle.Current models of the cross-bridge action in contracting muscle place the force-generating structural transition alternatively in the myosin head-rod junction [I, 21, in Conformational changes in the myosin molecule that might be relevant to the cross-bridge action have been extensively studied using a variety of experimental approaches (for review see [8 -1 I]). One of them involves examination of the effects of nucleotides and actin binding on the pattern and kinetics of proteolytic degradation of myosin and its subfragments. Limited digestion with trypsin of the chymotryptic subfragment 1 (Sl) of rabbit skeletal muscle myosin produces three heavy chain fragments, of 27 kDa, 50 kDa, and 20 kDa, representing the NH2-terminal, central, and COOH-terminal heavy chain domains, respectively. The binding of nucleotides to S1 results in the exposure of a new tryptic cleavage site in the NH2-terminal fragment, about 5 kDa from its NH2-terminus, and promotes an additional cleavage of the central fragment near its COOH-terminus [13 -171. These nucleotide- promoted cleavages are suppressed after formation of a ternary F-actin-S1 -nucleotide complex [15, 161. We have previously shown that the same site near the NH,-terminus is rendered susceptible to trypsin at low temperature in the absence of nucleotide, and that this low-temperature-promoted cleavage is also suppressed by actin. These data have been interpreted as indicating that the myosin heads exist as an equilibrium mixture of two conformational states even when they do not interact with nucleotide or actin [17]. Such a possibility has also been cons...

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Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion

Cited by 3 publications

References 10 publications

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy

New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

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Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion

Cited by 3 publications

References 10 publications

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by 1H NMR spectroscopy

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by 1H NMR spectroscopy

New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

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Product

Resources

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Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F‐actin studied by ¹H NMR spectroscopy