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. 1974 Mar;14(3):236–242. doi: 10.1016/S0006-3495(74)85911-4

Use of Fluorescence Polarization to Observe Changes in Attitude of S-1 Moieties in Muscle Fibers

T Nihei, Robert A Mendelson, Jean Botts
PMCID: PMC1334499  PMID: 4132695

Abstract

The fluorophore, N(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS), incubated with glycerinated psoas fibers primarily labels the S-1 moieties of such fibers, but it does not impair fiber contractility even when the degree of labeling is as high as 0.8 moles fluorophore per mole myosin. The polarization of the on-axis fluorescence from either the IAEDANS fluorophore, or the intrinsic tryptophane fluorophore, depends on whether the fiber is relaxed, in rigor, or developing isometric tension; furthermore, the changes in polarization on going from one state to another are much the same with either tryptophane or IAEDANS fluorophores. The foregoing is true whether the plane of the exciting light is parallel or perpendicular to the fiber axis. Also, if a fiber is first freed of its myosin by extraction, and is then incubated with IAEDANS-labeled S-1 the resulting polarization approaches that observed with a labeled, unextracted fiber in rigor. By contrast, incubation with the fluorophore, 7-nitro-4-chlorobenz-2-oxa-1,3-diazole (NBD-Cl) confers fluorescence only on actin, without impairing contractility, but the polarization of such fluorescence changes in a different direction and magnitude from myosin-originating fluorescence. It is concluded from these various observations that whether the fluorophore is IAEDANS or tryptophane the polarization change with change in physiological state originates in the S-1 moieties of fibers, and relates to the space attitude of these moieties.

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Selected References

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

  1. Aronson J. F., Morales M. F. Polarization of tryptophan fluorescence in muscle. Biochemistry. 1969 Nov;8(11):4517–4522. doi: 10.1021/bi00839a044. [DOI] [PubMed] [Google Scholar]
  2. Dos Remedios C. G., Millikan R. G., Morales M. F. Polarization of tryptophan fluorescence from single striated muscle fibers. A molecular probe of contractile state. J Gen Physiol. 1972 Jan;59(1):103–120. doi: 10.1085/jgp.59.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dos Remedios C. G., Yount R. G., Morales M. F. Individual states in the cycle of muscle contraction. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2542–2546. doi: 10.1073/pnas.69.9.2542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. HANSON J., HUXLEY H. E. Quantitative studies on the structure of cross-striated myofibrils. II. Investigations by biochemical techniques. Biochim Biophys Acta. 1957 Feb;23(2):250–260. doi: 10.1016/0006-3002(57)90326-8. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Huxley H. E., Brown W. The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J Mol Biol. 1967 Dec 14;30(2):383–434. doi: 10.1016/s0022-2836(67)80046-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Miller A., Tregear R. T. Structure of insect fibrillar flight muscle in the presence and absence of ATP. J Mol Biol. 1972 Sep 14;70(1):85–104. doi: 10.1016/0022-2836(72)90165-9. [DOI] [PubMed] [Google Scholar]
  9. Nihei T., Kay C. M. Isolation and properties of an enzymatically active fragment from papain-digested myosin. Biochim Biophys Acta. 1968 May 6;160(1):46–52. doi: 10.1016/0005-2795(68)90062-7. [DOI] [PubMed] [Google Scholar]
  10. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  11. Reedy M. K., Holmes K. C., Tregear R. T. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature. 1965 Sep 18;207(5003):1276–1280. doi: 10.1038/2071276a0. [DOI] [PubMed] [Google Scholar]
  12. Richards E. G., Chung C. S., Menzel D. B., Olcott H. S. Chromatography of myosin on diethylaminoethyl-Sephadex A-50. Biochemistry. 1967 Feb;6(2):528–540. doi: 10.1021/bi00854a022. [DOI] [PubMed] [Google Scholar]

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