Abstract
A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca2+-activated force. The results show that (1) about 50% of MLC2 were 32P-phosphorylated after activation of the skinned fibre preparation by 30 μM [Ca2+] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1–2 s after the sudden rise in [Ca2+] from below 10 nM to 30 μM; and (4) the sensitivity of the contractile apparatus to Ca2+ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca2+]. The K d for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 μM. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca2+ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Ashley CC, Moisescu DG (1975) The part played by Ca2+ in the contraction of isolated bundles of myofibrils. In: Carafoli E, et al. (eds) Calcium transport in contraction and secretion. North Holland, Amsterdam, pp 517–525
Blumenthal DK, Stull JT (1980) Activation of skeletal muscle myosin light chain kinase by calcium (2+) and calmodulin. Biochemistry 19:5608–5614
Cavadore JC, Molla A, Harricane M-C, Gabrion J, Benyamin Y, Demaille JG (1982) Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles. Proc Natl Acad Sci USA 79:3475–3479
Chisholm AAK, Cohen P (1988) The myosin-bound form of protein phosphatase 1 (PP-1M) is the enzyme that dephosphorylates native myosin in skeletal and cardiac muscles. Biochim Biophys Acta 971:163–169
Conti MA, Adelstein RS (1991) Purification and properties of myosin light chain kinases. Methods Enzymol 196:34–47
Crank J (1967) The mathematics of diffusion. Clarendon Press, Oxford
Edelman AM, Takio K, Blumenthal DK, Hansen RS, Walsh KA, Titani K, Krebs EG (1985) Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase. J Biol Chem 260:11 275–11 285
Edelman AM, Blumenthal DK, Krebs EG (1987) Protein serine/threonine kinases. Annu Rev Biochem 56:576–613
England P (1980) Protein phosphorylation in the regulation of muscle contraction. In: Cohen P (ed) Molecular aspects of cellular regulation, vol 1. Elsevier, Amsterdam, pp 153–173
Franco R, Rosenfeld MG (1990) Hormonally inducible phosphorylation of a nuclear pool of ribosomal protein S6. J Biol Chem 265:4321–4325
Giulian GG, Moss RL, Greaser ML (1983) Improved methodology for analysis and quantisation of proteins on one-dimensional silver-stained slab gels. Anal Biochem 129:277–287
Godt RE, Nosek TM (1989) Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J Physiol (Lond) 412:155–180
Kushmerick MJ, Podolsky RJ (1969) Ionic mobility in muscle cells. Science 166:1297–1298
Mayr GW, Heilmeyer MG (1983) Shape and substructure of skeletal muscle myosin light chain kinase. Biochemistry 22:4316–4326
Metzger JM, Greaser ML, Moss RL (1989) Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibres. J Gen Physiol 93:855–883
Moore R, Stull JT (1984) Myosin light chain phosphorylation in fast and slow skeletal muscles in situ. Am J Physiol 247:C462-C471
Moore RL, Palmer BM, Williams SL, Tanabe H, Grange RW, Houston ME (1990) Effect of temperature on myosin phosphorylation in mouse skeletal muscle. A J Physiol 259:C432-C438
Natori R (1954) The property and contraction process of isolated myofibrils. Jikeika Med J 1:119–126
Nunnaly MH, Rybicki SB, Stull JT (1985) Characterization of chicken skeletal muscle myosin light chain kinase. J Biol Chem 260:1020–1026
Payne MR, Rudnick SE (1989) Regulation of vertebrate striated muscle contraction. Trends Biochem Sci 14:357–360
Persechini A, Stull JT, Cooke R (1985) The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibres. J Biol Chem 260:7951–7954
Pires EM, Perry SV (1977) Purification and properties of myosin light-chain kinase from skeletal muscle. Biochem J 167:137–146
Salviati G, Betto R, Danielli Betto D (1982) Polymorphism of myofibrillar proteins in rabbit skeletal-muscle fibres. Biochem J 207:261–272
Shenolikar S, Nairn AC (1991) Protein phosphatases: recent progress. Adv Second Messengers Phosphoprotein Res 23:3–121
Stephenson DG (1988) Ca2+-activation of contraction in skeletal and cardiac muscle. Proc Austr Physiol Pharmacol Soc 19:108–122
Stephenson DG, Williams DA (1981) Calcium-activated force responses in fast and slow-twitch skinned muscle fibres of the rat at different temperatures. J Physiol (Lond) 317:281–302
Stephenson DG, Stewart AW, Wilson GJ (1989) Dissociation of force from myofibrillar MgATPase and stiffness at short sarcomere lengths in rat and toad skeletal muscle. J Physiol (Lond) 410:351–366
Stephenson GMM (1993) Studies of myosin light chain phosphorylation by the endogenous enzyme system in mechanically skinned muscle fibres of the rat. J Physiol (Lond) 459:17P
Stephenson GMM, Stephenson DG (1991) Simultaneous measurement of protein phosphorylation and contractile activity in mechanically skinned skeletal muscle fibres of the rat. Proc Austr Physiol Pharmacol Soc 22:70P
Stull JT, Sanford CF, Manning DR, Blumenthal D, High C (1981) Phosphorylation of myofibrillar proteins in striated muscles. Cold Spring Harbor Conf Cell Proliferation (Protein Phosphorylation) 18:823–839
Stull JT, Nunnaly MH, Moore RL, Blumenthal DK (1985) Myosin light chain kinases and myosin phosphorylation in skeletal muscle. Adv Enzyme Regul 23:123–140
Sweeney HL, Stull JT (1990) Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: Implications for regulation of actin-myosin interaction. Proc Natl Acad Sci USA 87:414–418
Takio K, Blumenthal DK, Walsh KA, Titani K, Krebs EG (1986) Amino acid sequence of rabbit skeletal muscle myosin light chain kinase. Biochemistry 25:8049–8057
Yates LD, Greaser ML (1983) Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol 168:123–141
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Stephenson, G.M.M., Stephenson, D.G. Endogenous MLC2 phosphorylation and Ca2+-activated force in mechanically skinned skeletal muscle fibres of the rat. Pflügers Arch. 424, 30–38 (1993). https://doi.org/10.1007/BF00375099
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00375099