M. Wick1
The Ohio State University Department of Animal Sciences
Meat is the edible muscle tissue of animals. The sarcomere is the fundamental functional unit of muscle. Growth and development of muscle is the result of the highly ordered accretion and assembly of the constituent proteins in the sarcomere. Primary amino acid sequence elements of the constitutive proteins carry the information necessary for determining the final architecture of the sarcomere. The mechanisms by which the constitutive proteins are assembled and function together to form the sarcomere and produce muscle contraction is just now beginning to be understood.
The predominant protein in the sarcomere, found in the thick filament system, is myosin. In physiological buffers, purified myosin spontaneously assembles into a synthetic thick filament with a dramatic resemblance to the native thick filament. Some of the amino-acid-sequence elements contributing to myosin’s assembly properties may also be critical to myosin’s solubility functions that are so crucial to the manufacture of high-quality prepared-meat products. This paper, written in August 1998, was presented to the symposium on cell biology at the 56th Annual Meeting of the Poultry Science Association, The Pennsylvania State University, and summarizes recent experimental results contributing to our understanding of the mechanism of sarcomeric muscle myosin assembly.
Striated muscle is found in all animal groups from coelenterates through vertebrates and comprises 80% or more of all muscular tissue (Hickmand, 1970). Muscle comprises nearly 40% of the body mass of most animals. Figure 1 diagrams the salient features of skeletal muscle cellular organization. Skeletal muscle is made of elongated cells or myofibers specialized for contraction. Each myofiber is approximately 100 µm thick and contains up to 1,000 myofibrils, each about 1 to 2 µm thick. In striated or skeletal muscle tissue, myofibrils display a pattern of alternating light (I) and dark (A) bands. The striations arise as a result of the packing arrangement of the filament systems in the sarcomere.
The sarcomere is the basic contractile unit of skeletal muscle and is defined as that portion of the myofibril between two Z-disks. Viewed in two dimensions, the sarcomere consists of two sets of filaments, thick and thin. Thin filaments are composed of filamentous actin (F-actin), each anchored at one end in perpendicularly aligned Z-disks. Associated with the thin filaments are the proteins of the contractile regulation system, tropomyosin (Tm) and troponin (TnI, TnT & TnC), and the giant proteins, titin and nebulin, reviewed elsewhere (Moos et al., 1995). Interdigitating between the thin filaments are bipolar thick filaments, composed almost entirely of the fibrous contractile protein myosin. The fine structure of the A-band and of the various myosin-binding and other filament-binding proteins of native thick filaments is reviewed elsewhere (Davis, 1988a; Sjostrom and Squire, 1977; Seiler et al., 1996).
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| Figure 1. Schematic relating the biochemical components to the microscopic structure of muscle tissue. (A) Cross section of muscle and (B) a longitudinal view of a muscle fiber showing the striated pattern of muscle cells. (C) The ultrastructure of the sarcomere. (D) Magnified view of thick and thin filaments and their associated filament system. |
Myosin
Sarcomeric myosins belong to the Type II class of myosin proteins that contain an a-helical coiled-coil rod domain that is involved in the assembly of bipolar thick native and synthetic filaments (Goodson and Spudich, 1993; Cheney et al., 1993). Myosin is a relatively large protein with a molecular mass of about 520 kDa (Figure 2). Each myosin molecule is composed of two 220 kDa heavy chains (MyHCs) and four light chains (LCs), ranging from 17 kDa to 22 kDa (Lowey and Risby, 1971). The entire molecule is approximately 160 nm in length and 2 nm in diameter. The heavy chains interact to form two distinct domains: a pair of globular heads (S1) 15 nm long and 9 nm wide and a-helical coiled-coil rod domain. The rod domain is approximately 150 nm long and 2 nm in diameter and is composed of the C-terminal 1100 amino acids of the MyHC. The myosin rod is a two- stranded coiled-coil motif characterized by two parallel amphipathic a-helical chains, which intertwine around each other into a left-handed superhelix (McLachlan and Karn, 1982; Crick, 1953).
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| Figure 2. Schematic representation of a sarcomeric myosin molecule. (A) Myosin, a 520-kDa hexameric molecule, is shown along with the common proteolytic fragments, (B) the low-salt soluble heavy meromyosin (HMM), and (C) the low-salt insoluble 130 kDa light meromyosin (LMM). |
Structure/function studies on myosin were greatly aided by the discovery that proteolytic enzymes, such as chymotrypsin, trypsin, and subtilisin, cleave myosin into well-defined, high molecular weight fragments (Mihalyi and Szent-Gyorgyi, 1953). Hydrodynamic and electron microscope studies demonstrated that trypsin, chymotrypsin, and subtilisin cleavage occurs predominantly within the rod domain approximately 80 nm from the C-terminus, generating two fragments. The larger fragment, termed heavy meromyosin (HMM), has a molecular weight of 140 kDa, is soluble in low ionic strength buffers, contains the S1 motor domain which is associated with the light chains, has ATPase activity, and binds actin. The 3-D structure of the S1 has recently been determined by X-ray crystallography. Based on this structure a major update in the mechanism of force generation has been proposed.
The smaller fragment, termed light meromyosin (LMM), has a molecular weight of 80 kDa, is composed of the C-terminal two-thirds of the rod domain, and confers the solubility and aggregation properties to the MyHC. The LMM has been shown to form paracrystalline structures at low ionic strength buffers (Strzelecka-Golaszewska et al., 1985; Harrison et al., 1971; Chowrashi and Pepe, 1977; Atkinson and Stewart, 1991b). Each mole of MyHC associates with 2 moles of light chains (Baba et al., 1984; Collins, 1976; Margossian et al., 1983).
MyHC Rod Domain
The information necessary for myosin’s assembly functions is carried in the unique repetitive primary sequence elements of the long a-helical coiled-coil rod domain. The rod consists of two right hand a-helical coils that intertwine around one another forming a left-handed coiled-coil characteristics of the side-chain residues of the amino acids in the primary sequence are repeated every 28 amino acids. Each repeat is composed of 4 seven-amino-acids clusters, called heptads, in which the side-chain residues are similar every seventh amino acid and are labeled a-b-c-d-e-f-g.
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| Figure 3. (A) Helical wheel representation of the a-helical coiled-coil motif of the myosin rod. The view is from the N-terminus of the rod domain the heptad positions labeled a through g. (B) Vertical representation of a-helical coiled-coil, 2 heptads. |
Fourier transform analysis of the charge characteristics of the primary sequence in each a-helix in the LMM has determined that each 28-residue repeat displays a characteristic sinusoidal pattern of alternating positive then negative charged residues in the b and c positions of every other heptad. The b and c positions in the first heptad in every repeat are predominantly occupied by lysine (K) or arginine (R) residues, whereas b and c positions in the third heptad are occupied by aspartic acid (D) or glutamic acid (E) residues. Thus, the typical repeat is arranged into alternating bands of positive and negative charges (McLachlan and Karn, 1982; McLachlan and Karn, 1983; Parry, 1981), leading to alternating positively and negatively charged zones arrayed along the outer surface of the entire rod (Figure 4).
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| Figure 4. Evolutionary conserved charge character in the LMM. The positions of evolutionary conserved positively charged amino-acid side-chain residues are represented by black rectangles, and negatively charged amino-acid side-chain residues by white rectangles. These clusters are arrayed on the solvent or outer surface of the LMM. The diagram is made by unwinding the a-helix in one of the coiled-coils. Each repeat (28 amino acids) is stacked individually like rungs on a ladder with the next C-terminal repeat aligned underneath. The numbers to the left indicate the repeat number in the a-helix. The LMM is composed of the C-terminal 22-1/2 repeats of the rod from repeat #17 to repeat #40. Each repeat is composed of four heptads, labeled at the top of the figure. Gray boxes indicate neutral, uncharged, or hydrophobic amino-acid side-chain residues. Hatched boxes indicate the position of skip residues in the LMM. Connected ovals at the bottom of the diagram represent the fact that these residues are in a random coil motif. |
The observation of this pattern has prompted some investigators to suggest that the reduction in the free energy by the neutralization of opposite charges between myosin rods could provide the energy necessary for myosin assembly if the molecules were staggered by odd multiples of 14 residues (Matsuda et al., 1982; McLachlan and Karn, 1982; McLachlan, 1983; Parry, 1981).
Synthetic Filaments
Myosin is extracted from myofibrils in salt solutions greater than 0.3 M. Purified myosin will precipitate by reducing the ionic strength of the salt solution to < 0.2 M and can be recovered by centrifugation at 5000 X g. If a solution of purified monomeric myosin in 0.6 M NaCl is negatively stained and examined by electron microscopy, no structures or particles are observed. The myosin monomer can be visualized in the electron microscope by the use of shadow-casting, a technique in which a thin carbon/platinum coating is sprayed on the structure (Rice, 1961).
As the ionic strength of a purified myosin solution is lowered from 0.6 M to less than 0.2 M NaCl, rod-shaped particles, termed synthetic filaments to distinguish them from native thick filaments, are easily viewed by transmission electron microscopy (TEM) after they have been embedded in a very thin block of uranium acetate. Synthetic filaments display most of the morphological characteristics of native thick filaments and are a good model to study the mechanism of myofibril-logenesis, myosin assembly, and the architecture of native thick filaments (Davis, 1985; Davis, 1988b; Huxley, 1963; Josephs and Harrington, 1966; Katsura and Noda, 1971; Pinset-Harstrom and Truffy, 1979; Pollard, 1975; Reisler et al., 1982).
A putative mechanism of synthetic filament assembly (Figure 5) has been proposed based on the studies of Reisler and Davis (Reisler et al., 1986; Davis, 1988b; Davis, 1986; Davis et al., 1982). Monomers initially assemble into parallel dimers. Dimers assemble into antiparallel tetramers, tetramers into octamers. The octamers finally assemble into a minifilament of 16 molecules, which is the nucleation core for additional assembly and corresponds to the central bare zone of the thick filament. Parallel dimers add on to the tips of the nascent filament in a bipolar fashion until the rate of addition of dimers equals the rate of dissociation of dimers, accounting for the narrow length distribution of synthetic filaments (Davis, 1981; Davis et al., 1982; Davis, 1985; Davis, 1986; Davis, 1988a). Based on these studies, the following assembly mechanism was proposed: as the ionic strength is lowered, myosin monomers interact to form parallel myosin dimers, which then associate to form a minifilament consisting of myosin associated in an antiparallel arrangement (Reisler et al., 1982; Reisler et al., 1986).
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| Figure 5. A putative mechanism of myosin assembly. (A) Myosin monomers in high salt spontaneously assemble into (B) parallel dimers, (C) minifilaments, and finally, (D) synthetic filaments as the ionic strength of the solution is reduced. |
Studies on the effects of anti-rod monoclonal antibody Fab fragments on sarcomeric muscle myosin interactions in low-salt conditions confirmed that regions in the C-terminus of the LMM were responsible for the solubility properties of chicken pectoralis major (PM) muscle myosin (Wick et al., 1997). Unique to these studies were the results that suggest the presence of domains in the N-terminus of the rod controlling the morphology of thick filaments and the architecture of the sarcomere.
The mechanism by which the length distribution of native and synthetic thick filaments is achieved is still a major question in muscle biology. The mechanism has to be consistent with the results of kinetic experiments in which a cumulative property progressively destabilizes the structure of the dimer binding site of the nascent filament. Thermodynamic insight into the mechanism arises from the observation in the electron microscope that native filaments split into three subfilaments in low-salt buffers (Huxley, 1963). The splitting is limited to the tips of the filament where myosin is parallel packed. The central bare zone appears to remain intact. The stable subfilaments in the central bare zone appear to be formed by strong attractive electrostatic interactions, whereas repulsive ionic interactions appear to exist in the regions of the filament in which splits occur. Since filaments formed at pH 8.0 are shorter than filaments formed at pH 7.0, it has been postulated that the repulsive interactions responsible for the splitting of native filaments in low salt are negative in charge (Davis, 1988 b).
Paracrystals
In order to determine the location and presence of amino acid sequence elements in the rod domain of the MyHC and their roles in the mechanism of fibrillogenesis, site-directed mutagenesis of recombinant proteins is becoming the tool of choice. However, a full-length functional hexameric recombinant sarcomeric muscle myosin molecule has yet to be produced in vitro. Therefore, investigations into the mechanisms of fibrillogenesis currently employ enzymatically generated and genetically engineered recombinant MyHC rod fragments.
Muscle and nonmuscle myosin rod fragments have been employed to study the intermolecular interactions responsible for filament assembly and solubility (Atkinson and Stewart, 1992; Chowrashi and Pepe, 1977; Chowrashi et al., 1989; Lee et al., 1994; O’Halloran et al., 1990; Ward and Bennett, 1989). Isolated LMM and rod fragments form paracrystals rather than filaments (Ward and Bennett, 1989; Szent-Gyorgyi et al., 1960; Stewart et al., 1989; Parry, 1981; Ishii and Lehrer, 1989; Chowrashi and Pepe, 1977; Atkinson and Stewart, 1991a). In buffers of low ionic strength, the light meromyosin (LMM) fragments of the rod assemble into highly ordered aggregates termed paracrystals, with axial banding patterns based on odd multiples of 14 nm believed to reflect the architecture of the arrangement of myosin in the thick filament (Figure 6). LMM fragments exhibit solubility characteristics that are indistinguishable from full-length myosin. Hence, LMM fragments have proved to be good tools to investigate the assembly properties of sarcomeric myosins. Deletions of the C-terminal portion of a bacterially expressed recombinant LMM generated a fragment that remained soluble in low salt (Atkinson and Stewart, 1991a; Sinard et al., 1989; Sinard et al., 1990). The contribution of the C-terminal 100 amino acids to the solubility and N-terminal sequence elements to the assembly of sarcomeric muscle myosin has been demonstrated with bacterially-expressed LMM fragments (Atkinson and Stewart, 1991a). The results of these studies indicated the possibility of sub-domains in the LMM that contribute to various low-energy states of low-salt aggregates of myosin and affect the morphology of native and synthetic filaments.
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| Figure 6. Myosin and rod fragments assemble into aggregates with different morphologies. (A) Illustrates a group of purified myosin molecules. (B) Transmission electron micrograph of a typical negatively stained synthetic filament. The central bare zone is visible in the center of a tapered bipolar aggregate approximately 1.6 mm in length and 15 nm in diameter. (C) LMM fragments are shown to assemble into paracrystals. (D) Transmission electron micrograph shows a typical negatively stained paracrystal. Paracrystals exhibit little or no length or width constraints. The bar = 0.5 mm. |
MyHC rod fragments assemble into para-crystals, rather than the precisely regulated synthetic filaments, with a narrow distribution of length and width. These observations led to the hypothesis that the bulkiness of the S1 domain of myosin may influence the assembly process. In support of this hypothesis, it was shown that removal of the LC2 from native myosin affected the morphology of synthetic filaments, implicating the myosin head in the mechanism of filament assembly (Chowrashi and Pepe, 1989). We are performing experiments employing bacterially expressed recombinant rod proteins in order to study the sequence elements in the N-terminus of the MyHC involved in myosin’s assembly properties. These studies will lead to the discovery of previously undescribed domains in the MyHC. Analysis of the sequence elements in these domains will contribute to our understanding of the intermolecular interactions occurring not only between individual myosin molecules that contribute to thick filament architecture but also between myosin molecules and with other constitutive myofibrillar proteins in determining the architecture of the sarcomere.
Atkinson, S. J. and Stewart, M. 1991a. Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation. J. Cell Sci. 99: 823—836.
Atkinson, S. J. and Stewart, M. 1991b. Molecular basis of myosin assembly: coiled-coil interactions and the role of charge periodicities. J.Cell Sci. Supplement 14, 7—10.
Atkinson, S. J. and Stewart, M. Molecular interactions in myosin assembly role of the 28-residue repeat in the rod. Mol. Biol. 1992, 226, 7—13.
Baba, M. L., Goodman, M., Berger-Cohn, J., Demaille, J. G., and Matsuda, G. The early adaptive evolution of calmodulin. Mol. Biol. Evol. 1984, 1, 442—455.
Bandman, E., Moore, L. A., Arrizubieta, M. J., Tidyman, W. E., Herman, L., and Wick, M. Soc. Gen. Physiol. Ser., Marine Biological Laboratory, Woods Hole, Mass.; The Rockefeller University Press, N.Y.; Vol. 49, p. 129—139.
Cheney, R. E., Riley, M. A., and Mooseker, M. S. Phylogenetic analysis of the myosin superfamily. Cell Motility and the Cytoskel. 1993, 24, 215—223.
Chowrashi, P. K., Pemrick, S. M., and Pepe, F. A. LC2 involvement in the assembly of skeletal myosin filaments. Biochinica et Biophysica Acta 1989, 990, 216—223.
Chowrashi, P. K. and Pepe, F. A. Light meromyosin paracrystal formation. J. Cell Biol. 1977, 74, 136—152.
Collins, J. Homology of myosin DTNB light chain with alkali light chains, troponin C and parvalbumin. Nature 1976, 259, 699—700.
Crick, F. H. C. The packing of a- helices: simple coiled-coils. Acta Cryst. 1953, 6, 689—697.
Davis, J. S. Pressure-jump studies on the length-regulation kinetics of the self-assembly of myosin from vertebrate skeletal muscle into thick filament. Biochem. J. 1981, 197, 309—314.
Davis, J. S. Kinetics and thermodynamics of the assembly of the parallel- and antiparallel-packed sections of synthetic thick filaments of skeletal myosin: a pressure-jump study. Biochem. 1985, 24, 5263—5269.
Davis, J. S. A model for length-regulation in thick filaments of vertebrate skeletal myosin. Biophys. J. 1986, 50, 417—422.
Davis, J. S. Assembly process in vertebrate skeletal thick filament formation. Annual Review of Biophysics and Biophysical Chem. 1988a, 17, 217—239.
Davis, J. S. Interaction of C-protein with pH 8.0 synthetic thick filaments prepared from the myosin of vertebrate skeletal muscle. J. Musc. Res. Cell Motil. 1988b, 9, 174—183.
Davis, J. S., Buck, J., and Greene, E. P. The myosin dimer: an intermediate in the self-assembly of the thick filament of vertebrate skeletal muscle. FEBS Lett 1982, 140, 293—297.
Goodson, H. V. and Spudich, J. A. Molecular evolution of the myosin family: Relationships derived from comparisons of amino acid sequences. Proc. Natl. Acad. Sci. (U.S.A.) 1993, 90, 659—663.
Harrison, R. G., Lowey, S., and Cohen, C. Assembly of myosin. J. Mol. Biol. 1971, 59, 531—535.
Hickmand, C. L. Integrated Principles of Zoology. 4th Ed. The C. V. Mosby Co., Saint Louis, Mo., 1970.
Huxley, H. E. Electron microscope studies on the structure of natural and synthetic filaments from striated muscle. Mol. Biol. 1963, 7, 281—308.
Ishii, Y. and Lehrer, S. Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon: effects of pH, salt, temperature, and troponin binding. Biophysical J. 1989, 56, 107—114.
Josephs, R. and Harrington, W. F. Studies on the formation and physical chemical properties of synthetic myosin filaments. Biochem. 1966, 11, 3474—3487.
Katsura, I. and Noda, H. Studies on the formation and physical chemical properties of synthetic myosin filaments. J. Biochem. 1971, 69, 219—229.
Lee, R. J., Egelhoff, T. T., Spudich, J. A. Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain. J. Cell Sci. 1994, 102, 2875—2886.
Lowey, S. and Risby, D. Light chains from fast and slow muscle myosins. Nature 1971, 234, 81-85.
Margossian, S. S., Bhan, A. K., and Slayter, H. S. Role of the regulatory light chains in skeletal muscle actomyosin ATPase and in minifilament formation. J. Biol. Chem. 1983, 258, 13359—13369.
Matsuda, R., Bandman, E., and Strohman, R. C. The two myosin isoenzymes of chicken anterior latissimus dorsi muscle contain different myosin heavy chains encoded by separate mRNAs. Differentiation 1982, 23, 36—41.
McLachlan, A. D. Analysis of gene duplication repeats in the myosin rod. J. Mol. Biol. 1983, 169, 15—30.
McLachlan, A. D., Karn, J. Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle. Nature 1982, 299, 226—231.
McLachlan, A. D. and Karn, J. Periodic features in the amino acid sequence of nematode myosin rod. J. Mol. Biol. 1983, 164, 605—626.
Mihalyi, E. and Szent-Gyorgyi, A. G. Trypsin digestion of muscle proteins. I. Ultracentrifugal analysis of the process. J. Biol. Chem. 1953, 201, 189—196.
Monera, O. D., Kay, C. M., Hodges, R. S. Electrostatic interactions control the parallel and antiparallel orientation of a-helical chains in two-stranded a-helical coiled-coils. Biochem. 1994, 33: 3862—3871.
Nguyen, H. T., Gubits, R. M., Wydro, R. M., Nadal-Ginard, B. Sarcomeric myosin heavy chain is coded by a highly conserved multigene family. Proc. Natl. Acad. Sci. (U.S.A.) 1982, 79, 5230—5234.
O’Halloran, T. J., Ravid, S., and Spudich, J. A. Expression of Dictyostelium myosin tail segments in Escherichia coli : Domains required for assembly and phosphorylation. J. Cell Biol. 1990, 110, 63—70.
Parry, D. A. Structure of rabbit skeletal myosin. Analysis of the amino acid sequences of two fragments from the rod region. J. Mol. Biol. 1981, 153, 459—464.
Pinset-Harstrom, I. and Truffy, J. Effect of adenosine triphosphate, inorganic phosphate and divalent cations on the size and structure of synthetic myosin filaments. Mol. Biol. 1979, 134, 173—188.
Pollard, T. D. Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge flexibility and coploymer formation. J. Cell Biol. 1975, 67, 93—104.
Reisler, E., Cheung, P., Borochov, N. Macromolecular assemblies of myosin. Biophys. J. 1986, 49, 335—342.
Reisler, E., Cheung, P., Oriol-Audit, C., and Lake, J. A. Growth of synthetic myosin filaments from myosin minifilaments. Biochem. 1982, 21, 701—707.
Rice, R. V. Conformation of individual macromolecular particles from myosin solutions. Biochimica et Biophysica Acta 1961, 52, 602—604.
Seiler, S. H., Fischman, D. A., Leinwand, L. A. Modulation of myosin filament organization by C-protein family members. Mol. Biol. Cell 1996, 7, 113-127.
Sinard, J. H., Rimm, D. L., and Pollard, T. D. Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. II. Assembly properties of tails with NH2- and COOH-terminal deletions. J. Cell Biol. 1990, 111, 2417—2426.
Sinard, J. H., Stafford, W. F., and Pollard, T. D. The mechanism of assembly by three successive dimerization steps. J. Cell Biol. 1989, 109, 1537-1547.
Sjostrom, M. and Squire, J. M. Fine structure of the A-band in cryo-sections: The structure of the A-band of human skeletal muscle fibers from ultra-thin cryo-sections negatively stained. J. Mol. Biol. 1977, 109, 49—68.
Stewart, M., Quinlan, R. A., Moir, R. D. Molecular interactions in paracrystals of a fragment corresponding to the a-helical coiled-coil rod portion of glial fibrillary acidic protein: Evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure. J. Cell Biol. 1989, 109, 225—34.
Strzelecka-Golaszewska, H., Nyitray, L., and Balint, M. Paracrystalline assemblies of light meromyosins with various chain weights. J. Musc. Res. Cell Motil. 1985, 6, 641—58.
Szent-Gyorgyi, A. G., Cohen, C., Philpott, D. E. Light meromyosin fraction I: A helical molecule from myosin. J. Mol. Biol. 1960, 2, 133—42.
Ward, R. and Bennett, P. Paracrystals of myosin rod. J. Mus. Res. Cell Motil. 1989, 10, 34—52.
Wick, M., Tablin, F., and Bandman, E. Effects of anti-LMM antibodies on the solubility of chicken skeletal muscle myosin. J. Food Biochem. 1997, 20, 379—95.
1 For more information, contact at: The Ohio State University 230A Plumb Hall, 2027 Coffey Road, Columbus, OH 43210, (614) 292-7516, Fax (614) 292-7116; email:wick.13@osu.edu