Summary
Tryptic digestion of myofibrils was used to assess the interaction of crossbridges with thin filaments in the presence of ATP analogues. The relative amounts of 200 kDa fragment produced by trypsin from myosin heavy chain when the crossbridge is attached to actin, and of 160 kDa fragment produced when the crossbridge is detached from actin, served as a measure of crossbridge-actin interaction. In rigor only the 200 kDa fragment was produced suggesting that a great majority of the crossbridges were strongly attached to actin; in the presence of MgPPi at 0° C only the 160 kDa fragment was finally produced suggesting that eventually all crossbridges detached from actin. In the presence of MgPPi or MgAMPPNP at 25° C both 200 and 160 kDa fragments were present for several minutes after myosin heavy chain had been completely digested, suggesting that two populations of crossbridges (attached and detached) co-existed at the same time within the myofibril. It is concluded that the addition of ATP analogues to muscle does not simply affect the chemical equilibrium of binding of myosin heads to actin but that it causes rapid dissociation of one crossbridge population without significant effect on binding to actin of the remaining crossbridge population.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J. &Sreter, F. A. (1978) Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin.Arch. Biochem. Biophys. 190, 793–9.
Borejdo, J. &Werber, M. M. (1982) Binding of calcium and magnesium to myosin in skeletal muscle myofibrils.Biochemistry 21, 549–55.
Borejdo, J., Assulin, O., Ando, T. &Putnam, S. (1982) Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore.J. molec. Biol. 158, 391–414.
Burghardt, T., Ando, T. &Borejdo, J. (1983) Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.Proc. natn. Acad. Sci. U.S.A. 80, 7515–19.
Chen, T. &Reisler, E. (1983) Tryptic digestion of myofibrils as a probe of myosin S-1 conformation.Biophys. J. 441, 102a.
Chen, T. &Reisler, E. (1984) Tryptic digestion of rabbit skeletal myofibrils: An enzymatic probe of myosin cross-bridge.Biochemistry 23, 2400–7.
Cooke, R. &Franks, K. (1980) All myosin heads form bonds with actin in rigor rabbit skeletal muscle.Biochemistry 19, 2265–9.
Cooke, R., Crowder, M. S. &Thomas, D. D. (1982) Orientation of spin labels attached to cross-bridges in contracting muscle fibres.Nature, Lond. 300, 776–8.
Goody, R. S., Holmes, K. C., Mannherz, H. G., Barrington Leigh, J. &Rosenbaum, G. (1975) Cross-bridge conformation as revealed by X-ray diffraction studies of insect flight muscles with ATP analogues.Biophys. J. 15, 687–705.
Highsmith, S. (1976) Interaction of the actin and nucleotide binding sites on myosin subfragment 1.J. biol. Chem. 251, 6170–2.
Hozumi, T. (1983) Structure and function of myosin subfragment 1 as studied by tryptic digestion.Biochemistry 22, 799–804.
Kuhn, H. J., Schroder, H. &Rüegg, J. C. (1972) Force generation in glycerinated insect flight muscles without ATP.Experientia 28, 510–11.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, Lond. 227, 680–5.
Lovell, S. J. &Harrington, W. F. (1981) Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor.J. molec. Biol. 149, 659–74.
Marston, S. B., Rodger, C. D. &Tregear, R. T. (1976) Changes in muscle cross-bridge when β, γ imido-ATP binds to myosin.J. molec. Biol. 104, 263–76.
Marston, S. B., Tregear, R. T., Rodger, C. D. &Clark, M. L. (1979) Coupling between the enzymatic site of myosin and the mechanical output of muscle.J. molec. Biol. 128, 111–26.
Matsuda, T. &Podolsky, R. J. (1984) X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.Biophys. J. 45, 9a.
Mornet, D., Pantel, P., Audemard, E. &Kassab, R. (1979) The limited tryptic cleavage of chymotryptic S-1: An approach to the characterization of the actin site in myosin heads.Biochem. Biophys. Res. Commun. 89, 925–32.
Reedy, M. C., Reedy, M. K. &Goody, R. S. (1983) Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibers fixed in rigor, in ATP and in AMPPNP.J. Musc. Res. Cell Motility 4, 55–81.
Schoenberg, M. &Eisenberg, E. (1984) Force decay following small stretches of freshly skinned rabbit psoas fibers in rigor PPi and AMPPNP solutions.Biophys. J. 45, 350a.
Spudich, J. P. &Watt, S. (1971) The regulation of rabbit skeletal muscle contraction.J. biol. Chem. 246, 4866–71.
Thomas, D. D. &Cooke, R. (1980) Orientation of spin-labeled myosin heads in glycerinated muscle fibers.Biophys. J. 32, 891–906.
Tonomura, Y., Appel, P. &Morales, M. F. (1966) On the molecular weight of myosin. II.Biochemistry 5, 515–21.
Tregear, R. T., Milch, J. R., Goody, R. S., Holmes, K. C. &Rodger, C. D. (1979) The uses of some novel X-ray diffraction techniques to study the effect of nucleotides on cross-bridges in insect flight muscle. InCross-Bridge Mechanism in Muscle Contraction (edited bySugi, H. andPollack, G. H.), pp. 407–23. Tokyo: University of Tokyo Press.
Weeds, A. G. &Pope, B. (1977) Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility.J. molec. Biol. 111, 129–57.
White, D. C. S. (1970) Rigor contraction and the effect of various phosphate compounds on glycerinated insect flight and vertebrate muscle.J. Physiol., Lond. 208, 583–605.
Yamamoto, K. &Sekine, T. (1979) Interaction of myosin Subfragment-1 with actin.J. Biochem. 6, 1855–62.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Assulin, O., Borejdo, J. & Flynn, C. Actin-attached and detached crossbridges in myofibrils: Segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain. J Muscle Res Cell Motil 7, 167–178 (1986). https://doi.org/10.1007/BF01753418
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01753418