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. 1991 Aug;60(2):352–359. doi: 10.1016/S0006-3495(91)82060-4

Molecular charge dominates the inhibition of actomyosin in skinned muscle fibers by SH1 peptides.

P B Chase 1, T W Beck 1, J Bursell 1, M J Kushmerick 1
PMCID: PMC1260071  PMID: 1912278

Abstract

It is not definitively known whether the highly conserved region of myosin heavy chain around SH1 (Cys 707) is part of the actin-binding site. We tested this possibility by assaying for competitive inhibition of maximum Ca-activated force production of skinned muscle fibers by synthetic peptides which had sequences derived from the SH1 region of myosin. Force was inhibited by a heptapeptide (IRICRKG) with an apparent K0.5 of about 4 mM. Unloaded shortening velocity of fibers, determined by the slack test, and maximum Ca-activated myofibrillar MgATPase activity were also inhibited by this peptide, but both required higher concentrations. We found that other cationic peptides also inhibited force in a manner that depended on the charge of the peptide; increasing the net positive charge of the peptide increased its efficacy. The inhibition was not significantly affected by altering solution ionic strength (100-200 mM). Disulfide bond formation was not involved in the inhibitory mechanism because a peptide with Thr substituted for Cys was inhibitory in the presence or absence of DTT. Our data demonstrate that the net charge was the predominant molecular characteristic correlated with the ability of peptides from this region of myosin heavy chain to inhibit force production. Thus, the hypothesis that the SH1 region of myosin is an essential part of the force-producing interaction with actin during the cross-bridge cycle (Eto, M., R. Suzuki, F. Morita, H. Kuwayama, N. Nishi, and S. Tokura., 1990, J. Biochem. 108:499-504; Keane et al., 1990, Nature (Lond.). 344:265-268) is not supported.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Audemard E., Bertrand R., Bonet A., Chaussepied P., Mornet D. Pathway for the communication between the ATPase and actin sites in myosin. J Muscle Res Cell Motil. 1988 Jun;9(3):197–218. doi: 10.1007/BF01773891. [DOI] [PubMed] [Google Scholar]
  2. Bertrand R., Chaussepied P., Kassab R., Boyer M., Roustan C., Benyamin Y. Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113. Biochemistry. 1988 Jul 26;27(15):5728–5736. doi: 10.1021/bi00415a050. [DOI] [PubMed] [Google Scholar]
  3. Bettache N., Bertrand R., Kassab R. Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6028–6032. doi: 10.1073/pnas.86.16.6028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bidlingmeyer B. A., Cohen S. A., Tarvin T. L. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr. 1984 Dec 7;336(1):93–104. doi: 10.1016/s0378-4347(00)85133-6. [DOI] [PubMed] [Google Scholar]
  5. Botts J., Thomason J. F., Morales M. F. On the origin and transmission of force in actomyosin subfragment 1. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2204–2208. doi: 10.1073/pnas.86.7.2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brenner B. Technique for stabilizing the striation pattern in maximally calcium-activated skinned rabbit psoas fibers. Biophys J. 1983 Jan;41(1):99–102. doi: 10.1016/S0006-3495(83)84411-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chase P. B., Kushmerick M. J. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys J. 1988 Jun;53(6):935–946. doi: 10.1016/S0006-3495(88)83174-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaussepied P., Kasprzak A. A. Change in the actin-myosin subfragment 1 interaction during actin polymerization. J Biol Chem. 1989 Dec 5;264(34):20752–20759. [PubMed] [Google Scholar]
  9. Chaussepied P., Morales M. F. Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7471–7475. doi: 10.1073/pnas.85.20.7471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dos Remedios C. G., Cooke R. Fluorescence energy transfer between probes on actin and probes on myosin. Biochim Biophys Acta. 1984 Jul 31;788(2):193–205. doi: 10.1016/0167-4838(84)90262-0. [DOI] [PubMed] [Google Scholar]
  11. Eldin P., Le Cunff M., Diederich K. W., Jaenicke T., Cornillon B., Mornet D., Vosberg H. P., Léger J. J. Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin. J Muscle Res Cell Motil. 1990 Oct;11(5):378–391. doi: 10.1007/BF01739759. [DOI] [PubMed] [Google Scholar]
  12. Elzinga M., Collins J. H. Amino acid sequence of a myosin fragment that contains SH-1, SH-2, and Ntau-methylhistidine. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4281–4284. doi: 10.1073/pnas.74.10.4281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eto M., Suzuki R., Morita F., Kuwayama H., Nishi N., Tokura S. Roles of the amino acid side chains in the actin-binding S-site of myosin heavy chain. J Biochem. 1990 Sep;108(3):499–504. doi: 10.1093/oxfordjournals.jbchem.a123228. [DOI] [PubMed] [Google Scholar]
  14. Fryer M. W., Neering I. R., Stephenson D. G. Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibres. J Physiol. 1988 Dec;407:53–75. doi: 10.1113/jphysiol.1988.sp017403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldman Y. E., Simmons R. M. Control of sarcomere length in skinned muscle fibres of Rana temporaria during mechanical transients. J Physiol. 1984 May;350:497–518. doi: 10.1113/jphysiol.1984.sp015215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gordon A. M., Godt R. E., Donaldson S. K., Harris C. E. Tension in skinned frog muscle fibers in solutions of varying ionic strength and neutral salt composition. J Gen Physiol. 1973 Nov;62(5):550–574. doi: 10.1085/jgp.62.5.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huxley A. F., Simmons R. M. Proposed mechanism of force generation in striated muscle. Nature. 1971 Oct 22;233(5321):533–538. doi: 10.1038/233533a0. [DOI] [PubMed] [Google Scholar]
  18. Kasprzak A. A., Chaussepied P., Morales M. F. Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex. Biochemistry. 1989 Nov 14;28(23):9230–9238. doi: 10.1021/bi00449a040. [DOI] [PubMed] [Google Scholar]
  19. Katoh T., Imae S., Morita F. Binding of F-actin to a region between SH1 and SH2 groups of myosin subfragment-1 which may determine the high affinity of acto-subfragment-1 complex at rigor. J Biochem. 1984 Feb;95(2):447–454. doi: 10.1093/oxfordjournals.jbchem.a134626. [DOI] [PubMed] [Google Scholar]
  20. Katoh T., Katoh H., Morita F. Actin-binding peptide obtained by the cyanogen bromide cleavage of the 20-kDa fragment of myosin subfragment-1. J Biol Chem. 1985 Jun 10;260(11):6723–6727. [PubMed] [Google Scholar]
  21. Katoh T., Morita F. Interaction between myosin and F-actin. Correlation with actin-binding sites on subfragment-1. J Biochem. 1984 Oct;96(4):1223–1230. doi: 10.1093/oxfordjournals.jbchem.a134940. [DOI] [PubMed] [Google Scholar]
  22. Keane A. M., Trayer I. P., Levine B. A., Zeugner C., Ruegg J. C. Peptide mimetics of an actin-binding site on myosin span two functional domains on actin. Nature. 1990 Mar 15;344(6263):265–268. doi: 10.1038/344265a0. [DOI] [PubMed] [Google Scholar]
  23. Knight P. J., Trinick J. A. Preparation of myofibrils. Methods Enzymol. 1982;85(Pt B):9–12. doi: 10.1016/0076-6879(82)85004-0. [DOI] [PubMed] [Google Scholar]
  24. Mornet D., Bertrand R. U., Pantel P., Audemard E., Kassab R. Proteolytic approach to structure and function of actin recognition site in myosin heads. Biochemistry. 1981 Apr 14;20(8):2110–2120. doi: 10.1021/bi00511a007. [DOI] [PubMed] [Google Scholar]
  25. Mornet D., Bertrand R., Pantel P., Audemard E., Kassab R. Structure of the actin-myosin interface. Nature. 1981 Jul 23;292(5821):301–306. doi: 10.1038/292301a0. [DOI] [PubMed] [Google Scholar]
  26. Mornet D., Bonet A., Audemard E., Bonicel J. Functional sequences of the myosin head. J Muscle Res Cell Motil. 1989 Feb;10(1):10–24. doi: 10.1007/BF01739853. [DOI] [PubMed] [Google Scholar]
  27. Stafford W. F., 3rd Effect of various anions on the stability of the coiled coil of skeletal muscle myosin. Biochemistry. 1985 Jun 18;24(13):3314–3321. doi: 10.1021/bi00334a036. [DOI] [PubMed] [Google Scholar]
  28. Stedman H. H., Eller M., Jullian E. H., Fertels S. H., Sarkar S., Sylvester J. E., Kelly A. M., Rubinstein N. A. The human embryonic myosin heavy chain. Complete primary structure reveals evolutionary relationships with other developmental isoforms. J Biol Chem. 1990 Feb 25;265(6):3568–3576. [PubMed] [Google Scholar]
  29. Sutoh K. An actin-binding site on the 20K fragment of myosin subfragment 1. Biochemistry. 1982 Sep 14;21(19):4800–4804. doi: 10.1021/bi00262a043. [DOI] [PubMed] [Google Scholar]
  30. Sutoh K. Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry. 1983 Mar 29;22(7):1579–1585. doi: 10.1021/bi00276a009. [DOI] [PubMed] [Google Scholar]
  31. Suzuki R., Morita F. Actin-binding site of pig cardiac myosin. Biochim Biophys Acta. 1987 Mar 18;912(1):147–150. doi: 10.1016/0167-4838(87)90259-7. [DOI] [PubMed] [Google Scholar]
  32. Suzuki R., Morita F., Nishi N., Tokura S. Inhibition of actomyosin subfragment 1 ATPase activity by analog peptides of the actin-binding site around the Cys(SH1) of myosin heavy chain. J Biol Chem. 1990 Mar 25;265(9):4939–4943. [PubMed] [Google Scholar]
  33. Suzuki R., Nishi N., Tokura S., Morita F. F-actin-binding synthetic heptapeptide having the amino acid sequence around the SH1 cysteinyl residue of myosin. J Biol Chem. 1987 Aug 25;262(24):11410–11412. [PubMed] [Google Scholar]
  34. Sweeney H. L., Corteselli S. A., Kushmerick M. J. Measurements on permeabilized skeletal muscle fibers during continuous activation. Am J Physiol. 1987 May;252(5 Pt 1):C575–C580. doi: 10.1152/ajpcell.1987.252.5.C575. [DOI] [PubMed] [Google Scholar]
  35. Takashi R. Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1. Biochemistry. 1979 Nov 13;18(23):5164–5169. doi: 10.1021/bi00590a021. [DOI] [PubMed] [Google Scholar]
  36. Takashi R., Kasprzak A. A. Measurement of interprotein distances in the acto-subfragment 1 rigor complex. Biochemistry. 1987 Nov 17;26(23):7471–7477. doi: 10.1021/bi00397a041. [DOI] [PubMed] [Google Scholar]
  37. Thomas D. D. Spectroscopic probes of muscle cross-bridge rotation. Annu Rev Physiol. 1987;49:691–709. doi: 10.1146/annurev.ph.49.030187.003355. [DOI] [PubMed] [Google Scholar]
  38. Tokunaga M., Sutoh K., Toyoshima C., Wakabayashi T. Location of the ATPase site of myosin determined by three-dimensional electron microscopy. Nature. 1987 Oct 15;329(6140):635–638. doi: 10.1038/329635a0. [DOI] [PubMed] [Google Scholar]
  39. Trayer H. R., Trayer I. P. Fluorescence energy transfer between the myosin subfragment-1 isoenzymes and F-actin in the absence and presence of nucleotides. Eur J Biochem. 1983 Sep 1;135(1):47–59. doi: 10.1111/j.1432-1033.1983.tb07616.x. [DOI] [PubMed] [Google Scholar]
  40. Warrick H. M., De Lozanne A., Leinwand L. A., Spudich J. A. Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9433–9437. doi: 10.1073/pnas.83.24.9433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  42. White H. D. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol. 1982;85(Pt B):698–708. doi: 10.1016/0076-6879(82)85057-x. [DOI] [PubMed] [Google Scholar]
  43. Yates L. D., Greaser M. L. Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol. 1983 Jul 25;168(1):123–141. doi: 10.1016/s0022-2836(83)80326-x. [DOI] [PubMed] [Google Scholar]

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