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. 1985 Dec;82(24):8478–8482. doi: 10.1073/pnas.82.24.8478

Fraction of myosin cross-bridges bound to actin in active muscle fibers: estimation by fluorescence anisotropy measurements.

T P Burghardt, K Ajtai
PMCID: PMC390939  PMID: 3866235

Abstract

Time-resolved and steady-state fluorescence anisotropy measurements from fluorescence-labeled myosin cross-bridges in single glycerinated skeletal muscle fibers in rigor, relaxed, MgADP-induced, and contracting states have been made in order to estimate the fraction of actin-bound cross-bridges in active muscle. When the plane of polarization of the excitation light is perpendicular to the fiber axis and its propagation vector has a component parallel to this axis, actin-bound cross-bridge states, such as rigor and MgADP-induced, have time-zero and steady-state anisotropies that are substantially lower than has the relaxed state. This difference provides a means of determining the fraction of cross-bridges bound to actin in active isometric fibers, by comparing the fluorescence anisotropy from active fibers with the anisotropy from bound and unbound cross-bridges in static states. By assuming that the active cross-bridges are either bound (in the manner of rigor or MgADP-induced states) or relaxed, we estimate that greater than 80% of the cross-bridges are actin-bound in active isometric fibers.

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Selected References

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

  1. Badea M. G., Brand L. Time-resolved fluorescence measurements. Methods Enzymol. 1979;61:378–425. doi: 10.1016/0076-6879(79)61019-4. [DOI] [PubMed] [Google Scholar]
  2. Borejdo J., Assulin O., Ando T., Putnam S. Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore. J Mol Biol. 1982 Jul 5;158(3):391–414. doi: 10.1016/0022-2836(82)90205-4. [DOI] [PubMed] [Google Scholar]
  3. Borejdo J., Putnam S., Morales M. F. Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6346–6350. doi: 10.1073/pnas.76.12.6346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borejdo J., Putnam S. Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation. Biochim Biophys Acta. 1977 Mar 11;459(3):578–595. doi: 10.1016/0005-2728(77)90056-1. [DOI] [PubMed] [Google Scholar]
  5. Burghardt T. P., Ando T., Borejdo J. Evidence for cross-bridge order in contraction of glycerinated skeletal muscle. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7515–7519. doi: 10.1073/pnas.80.24.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burghardt T. P., Thompson N. L. Motion of myosin cross-bridges in skeletal muscle fibers studied by time-resolved fluorescence anisotropy decay. Biochemistry. 1985 Jul 2;24(14):3731–3735. doi: 10.1021/bi00335a048. [DOI] [PubMed] [Google Scholar]
  7. Burghardt T. P. Time-resolved fluorescence polarization from ordered biological assemblies. Biophys J. 1985 Oct;48(4):623–631. doi: 10.1016/S0006-3495(85)83818-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooke R., Crowder M. S., Thomas D. D. Orientation of spin labels attached to cross-bridges in contracting muscle fibres. Nature. 1982 Dec 23;300(5894):776–778. doi: 10.1038/300776a0. [DOI] [PubMed] [Google Scholar]
  9. Grinvald A., Steinberg I. Z. On the analysis of fluorescence decay kinetics by the method of least-squares. Anal Biochem. 1974 Jun;59(2):583–598. doi: 10.1016/0003-2697(74)90312-1. [DOI] [PubMed] [Google Scholar]
  10. Hudson E. N., Weber G. Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry. 1973 Oct 9;12(21):4154–4161. doi: 10.1021/bi00745a019. [DOI] [PubMed] [Google Scholar]
  11. Huxley H. E., Simmons R. M., Faruqi A. R., Kress M., Bordas J., Koch M. H. Millisecond time-resolved changes in x-ray reflections from contracting muscle during rapid mechanical transients, recorded using synchrotron radiation. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2297–2301. doi: 10.1073/pnas.78.4.2297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Matsubara I., Yagi N., Hashizume H. Use of an X-ray television for diffraction of the frog striated muscle. Nature. 1975 Jun 26;255(5511):728–729. doi: 10.1038/255728a0. [DOI] [PubMed] [Google Scholar]
  13. Matsubara I., Yagi N., Miura H., Ozeki M., Izumi T. Intensification of the 5.9-nm actin layer line in contracting muscle. 1984 Nov 29-Dec 5Nature. 312(5993):471–473. doi: 10.1038/312471a0. [DOI] [PubMed] [Google Scholar]
  14. Mendelson R. A., Morales M. F., Botts J. Segmental flexibility of the S-1 moiety of myosin. Biochemistry. 1973 Jun 5;12(12):2250–2255. doi: 10.1021/bi00736a011. [DOI] [PubMed] [Google Scholar]
  15. Nihei T., Mendelson R. A., Botts J. Use of fluorescence polarization to observe changes in attitude of S-1 moieties in muscle fibers. Biophys J. 1974 Mar;14(3):236–242. doi: 10.1016/S0006-3495(74)85911-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Takashi R., Duke J., Ue K., Morales M. F. Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS. Arch Biochem Biophys. 1976 Jul;175(1):279–283. doi: 10.1016/0003-9861(76)90509-9. [DOI] [PubMed] [Google Scholar]
  17. Torgerson P. M. Tryptophan emission from myosin subfragment 1: acrylamide and nucleotide effect monitored by decay-associated spectra. Biochemistry. 1984 Jun 19;23(13):3002–3007. doi: 10.1021/bi00308a024. [DOI] [PubMed] [Google Scholar]
  18. Yanagida T., Nakase M., Nishiyama K., Oosawa F. Direct observation of motion of single F-actin filaments in the presence of myosin. Nature. 1984 Jan 5;307(5946):58–60. doi: 10.1038/307058a0. [DOI] [PubMed] [Google Scholar]

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