Production of specific antibodies to contractile proteins,and their use in immunofluorescence microscopy

Summary Antibodies prepared against actomyosins can be shown to behave similarly, if not identically to more recently prepared antibodies against highly purified myosins. Details of the purification of the antigens, and of the production of antibodies to chick myosins from smooth gizzard muscle and from striated pectoral muscle are given. The antibody specificity appears to be directed against the heavy chains of the myosin molecules, since these antibodies specifically inhibit the myosin ATPase reaction, and sincein situ staining of myosin polypeptide chains on an SDS gel using the antibodies in indirect fluorescence shows staining only in the heavy band region. Use of the antibodies in immunofluorescence microscopy suggest that the antibodies are tissue, but not species, specific. Example of their use in staining tissue sections are shown.     

Purification of messenger ribonucleic acids for fast and slow myosin heavy chains by indirect immunoprecipitation of polysomes from embryonic chick skeletal muscle

Fast and slow myosin heavy chain mRNAs were isolated by indirect immunoprecipitation of polysomes from 14-day-old embryonic chick leg muscle. The antibodies were prepared against myosin heavy chains purified by NaDod-SO4-polyacrylamide gel electrophoresis and were shown to be specific for fast and slow myosin heavy chains. The RNA fractions directed the synthesis of myosin heavy chains in a cell-free translation system from wheat germ. Several smaller peptides were also synthesized in lower concentrations. These probably are partial products of myosin heavy chains, since they are immunoprecipitated with antibodies to myosin heavy chains. Immunoprecipitation of the translation products with the antibodies to fast and slow myosin heavy chains showed the RNA preparations to be approximately 94% enriched for fast myosin heavy chain mRNA and approximately 84% enriched for slow myosin heavy chain mRNA with respect to myosin HC type. Peptides having slightly different mobilities on NaDodSO4-polyacrylamide gels were immunoprecipitated by antibodies to fast and slow myosin heavy chains.     

Preparation of type-specific antimyosin antibodies and determination of their specificity by biochemical and immunohistochemical methods

This paper reports the preparation of specific anti-slow myosin antibodies (anti-I) and anti-fast myosin antibodies (anti-IIA) raised against myosins from sheep and guinea pig masseter muscles. The specificity of the antibodies has been studied by immunodiffusion in agar and by the GEDELISA test using slow-twitch (type I), fast-twitch red (type IIA) and fast-twitch white (type IIB) myofibrils isolated from guinea pig muscles. The principal specificity of the anti-I and anti-IIA antibodies was for the heavy chains of type I and IIA myosins, respectively. A smaller reaction with the corresponding light chains was also detected. Immunohistochemical staining of muscle sections using these antibodies confirmed their fibre type specificity.     

Myosin subunit composition in human developing muscle.

Previous pyrophosphate-gel studies have reported the existence of embryonic neonatal myosin isoenzymes in human developing muscle. The present investigation was undertaken to characterize their subunit composition more precisely. Two immature muscle myosins are contrasted with adult myosin: neonatal myosin and foetal myosin. The neonatal form of myosin is weakly cross-reactive with rabbit slow myosin and contains only fast-type light chains (LC), LC1F and LC2F. The associated heavy chains consist of a single electrophoretic component that reacts exclusively with antibodies against human foetal myosin and has a mobility and peptide pattern distinct from that of adult fast and slow heavy chains. Foetal myosin is distinguished by the presence of low amounts of a heavy chain immunologically cross-reactive with the adult slow form and of two additional light-chain components: a LC2S light chain and a foetal-specific light chain (LCemb.). The foetal-specific light chain, as shown by one-dimensional-peptide-map analysis, is structurally unrelated to both LC1S and LC1F light chains of human adult myosin. We conclude from these results that the ontogenesis of human muscle myosin shares certain common features with that observed in other species, except for the persistence until birth of a foetal form of heavy chain (HCemb.).     

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.     

Comparative analysis of chicken atrial and ventricular myosins.

1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2+-ATPase activity, both in function of pH and [K+], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two-dimensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.     

Regulation of protein synthesis and accumulation during oogenesis in Xenopus laevis

The tissue and developmental distribution of the various myosin subunits has been examined in bovine cardiac muscle. Electrophoretic analysis shows that a myosin light chain found in fetal but not in adult ventricular myosin is very similar and possibly identical to the light chain found in fetal or adult atrial and adult Purkinje fiber myosins. This light chain comigrates on two-dimensional gels with the bovine skeletal muscle embryonic light chain. Thus, this protein appears to be expressed only at early developmental stages in some tissues (cardiac ventricles, skeletal muscle) but at all stages in others (cardiac atria). The heavy chains of these myosins have been examined by one- and two-dimensional polypeptide mapping. The ventricular and Purkinje fiber heavy chains are indistinguishable. They are, however, different from the heavy chain found in cultured skeletal muscle myotubes, in contrast to the situation concerning the embryonic/atrial light chain.     

Purification and characterization of myosins from human and rabbit skeletal muscles by using specific monoclonal antibodies

By using immunoaffinity column chromatography slow (I) and fast (IIA, IIB) myosins were isolated from human (vastus lateralis) and rabbit (tibialis anterior, psoas and conoidal bundle) skeletal muscles. The peptide pattern revealed that slow (I) and fast (IIA, IIB) myosin heavy chains are quite distinct, as are those from pure slow (conoidal bundle) and fast (psoas) rabbit skeletal muscles. Unlike Billeter et al. (1981) the authors observed that fast human myosins were always associated with a small amount of slow myosin light chains. The fast myosins (IIA, IIB) from rabbit tibialis anterior muscle did not appear very distinct and contained only fast myosin light chains. These myosins were different from the IIB myosin from the psoas muscle. Ten per cent of the fibres revealed histochemically as fast IIA also reacted with an anti-slow myosin antibody. The classical histochemical techniques appear inadequate to demonstrate the existing differences among fibre types, but the monoclonal antibodies hold promise.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.     

Myosins exist as homodimers of heavy chains: demonstration with specific antibody purified by nematode mutant myosin affinity chromatography.

The body-walls of Caenorhabditis elegans contain two different myosin heavy chains (Epstein, Waterston and Brenner, 1974) that associate to form at least two species of myosin (Schachat, Harris and Epstein, 1977a). To better define the distribution of these heavy chains in myosin molecules, we have characterized the myosin of C. elegans by immunochemical methods. Specific, precipitating anti-myosin antibody has been prepared in rabbits using highly purified nematode myosin as the immunogen. The difference in reactivity of the anti-myosin antibody with wild-type myosin containing both kinds of heavy chains (designated unc-54 and non-unc-54 heavy chains on the basis of genetic specification) and myosin from the mutant E190 that lacks unc-54 heavy chains Indicates that there are antigenic differences between myosin molecules containing unc-54 heavy chains and myosin molecules containing only non-unc-54 heavy chains. Antibody specific for the unc-54 myosin determinants has been prepared by the immunoadsorption of anti-myosin antibody with E190 myosin. This specific anti-unc-54 myosin antibody precipitates myosin that contains only unc-54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, we could detect no heterodimeric myosin molecules containing both unc-54 and non-unc-54 heavy chains. The body-wall myosins of C. elegans therefore exist only as homodimers of either class of heavy chain.This specific anti-unc-54 myosin antibody promises to be a valuable tool in elucidating the role of two myosins in body-wall muscle and in molecular characterizations of mutant myosins in C. elegans. We report here the use of this antibody to detect antigenic differences between unc-54 myosin from the wild-type and the muscle mutant E675. In conjunction with the original anti-myosin antibody, other studies show that both unc-54 and non-unc-54 myosins exist within the same body-wall muscle cells (Mackenzie, Schachat and Epstein, 1978) and that both myosins are coordinately synthesized during muscle development in C. elegans (Garcea, Schachat and Epstein, 1978). We discuss the implications of the self-association of unc-54 and non-unc-54 myosin heavy chains into homodimeric myosins within the same body-wall muscles with respect to the assembly of thick filaments and their organization into a regular lattice.     

Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.     

Myosins of secretory tissues

Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development

Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage-specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.