Isolation and characterization of the three light chains from carp white muscle myosin.

Myosin from carp white muscle contains two mol of "DTNB" light chain (mol. wt 17 500 daltons) and two mol of "alkali" light chains (mol. wt 25 000 and 16 400 daltons). The three light chains have been isolated in pure state and characterized by electrofocusing, ultraviolet absorption, amino acid analysis and tryptic peptide mapping. Our results show a great homology between the two carp alkali light chains whereas LC2 seems chemically more different. But the homology of LC1 and LC3 is not so extensive as in the case of the higher vertebrate myosin.     

Mapping of fish myosin light chains by two-dimensional gel electrophoresis

1. Myosins were prepared from the ordinary muscle of 16 fish species as well as from rabbit fast muscle, and light chain subunits [alkali light chains A1, A2 and DTNB (5,5'-dithio-bis-2-nitrobenzoate) light chain] were separated on two-dimensional gel electrophoresis in combination with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. A1 light chains showed mol. wts ranging from 21,000 to 22,900 and isoelectric points ranging from 4.51 to 4.62. DTNB light chains were spotted in a narrow area, with a mol. wt range of 16,800-17,600 and an isoelectric point range of 4.48-4.55. On the other hand, A2 light chains were most species-specific, with a mol. wt range of 14,000-19,500 and an isoelectric point range of 4.31-4.46. 3. It was suggested that the lower species-specificity in A1 as opposed to A2 is accounted for by the addition of an N-terminal peptide ("difference peptide") in the former. The properties and possible role of this peptide are discussed.     

Immunochemical specificity of myosin light chains from mackerel ordinary and dark muscles

Five light chains were isolated from the ordinary and dark muscle myosins of mackerel Pneumatophorus japonicus japonicus, by a method consisting of DTNB and urea treatments, followed by DEAE-cellulose chromatography. Some physicochemical and immunochemical properties of the light chains thus obtained were analyzed. A1, A2, and DTNB light chains from ordinary muscle myosin resembled one another in ultraviolet absorption spectrum, as did D1 and D2 light chains from dark muscle myosin. However, the absorption spectra of the former three differed from those of the latter two. Amino acid compositions of A1 and A2 light chains resembled each other, except for a few amino acids such as lysine, proline, and alanine. Tryptophan was detected only in DTNB light chain. D1 and D2 light chains showed general similarity, except for a remarkably higher proline content in D1. Anti-A1 (or anti-A2) antiserum exhibited a cross-reaction against A2 (or A1) in both immunoelectrophoresis and ELISA, indicating an immunochemical similarity of these two alkali light chains. No precipitin line appeared when anti-A1 or anti-A2 antiserum was diffused against DTNB light chain in immunoelectrophoresis. In ELISA, however, each pair showed cross-reactivity values as high as 50-80%, values which were rather higher than those obtained with heterologous alkali light chains (10-40%). Anti-DTNB light chain antiserum reacted with either alkali light chain in both methods. Anti-D1 antiserum cross-reacted against D2, and anti-D2 antiserum did against D1. These myosin light chains exhibited a high immunochemical tissue-specificity.     

Lobster (Homarus americanus) striated muscle myosin.

1. Myosin from the thin-filament regulated flexor muscle of lobster contains 2 moles of each of 2 light chains. 2. The Lb 1 light chain of 19,000 daltons which can be removed by DTNB is heavier than the DTNB light chain of chicken. The Lb 2 light chain of 17,000 daltons can be removed with urea. 3. On electrophoresis in 8 M urea (pH 8.7) the Lb 2 light chain migrates with a mobility similar to that of chicken A2, but the Lb 1 migrates significantly faster than any of the chicken light chains. 4. In lobster, the DTNB treatment destroys the Ca and K-EDTA ATPase activity of lobster myosin.     

On the location of the divalent metal binding sites and the light chain subunits of vertebrate myosin.

The divalent metal ion binding sites of skeletal myosin were investigated by electron paramagnetic resonance (EPR) spectroscopy using the paramagnetic (Mn(II) ion as a probe. Myosin possesses two high affinity sites (K less than 1 muM) for Mn(II), which are located on the 5,5'-dithiobis(2-nitrobenzoate) (DTNB) light chains. Mn(II) bound to the isolated DTNB light chain gives rise to an EPR spectrum similar to that of Mn(II) bound to myosin and this indicates that the metal binding site comprises ligands from the DTNB light chain alone. Myosin preparations in which the DTNB light chain content is reduced by treatment with 5,5'-dithiobis(2-nitrobenzoate) show a corresponding reduction in the stoichiometry of Mn(II) binding, but the stoichiometry is recovered on reassociation of the DTNB light chain. Chymotryptic digestion of myosin filaments in the presence of ethylenediaminetetraacetic acid yields subfragment 1, but digestion in the presence of divalent metal ions produces heavy meromyosin. Myosin with a depleted DTNB light chain content gives rise to subfragment 1 on proteolysis, even in the presence of divalent metal ions. It is proposed that saturation of the DTNB light chain site with divalent ions protects this subunit against proteolysis, which, in turn, inhibits the cleavage of the subfragment 1-subfragment 2 link. Either the DTNB light chain is located near the region of the link and sterically blocks chymotryptic attack, or it is bound to the subfragment 1 moiety and affects the conformation of the link region. When the product heavy meromyosin was examined by sodium dodecyl sulfate gel electrophoresis, an apparent anomaly arose in that there was no trace of the 19 000-dalton band corresponding to the DTNB light chain. This was resolved by following the time course of chymotryptic digestion of the myosin heavy chain, the DTNB light chain, and the divalent metal binding site. The 19 000-dalton DTNB light chain is rapidly degraded to a 17 000-dalton fragment which comigrates with the alkali 2 light chain. The divalent metal site remains intact, despite this degradation, and the 17 000 fragment continues to protect the subfragment 1-subfragment 2 link. In the absence of divalent metal ions, the 17 000-dalton fragment is further degraded and attack of the subfragment 1 link ensues. Mn(II) bound to cardiac myosin gives an EPR spectrum basically similar to that of skeletal myosin, suggesting that their 19 000-dalton light chains are analogous with respect to their divalent metal binding sites, despite their chemical differences. The potential of EPR spectroscopy for characterizing the metal binding sites of myosin from different sources and of intact muscle fibers is discussed.     

Chicken secretory immunoglobulin: chemical and immunological characterization of chicken IgA.

1. Chicken IgA purified from biliary fluids was chemically and immunologically characterized. 2. Chicken IgA was determined to be the only immunoglobulin class present in bile. Gel filtration studies reveal polymeric IgA e.g. 17-19S. 3. Antigenically, chicken IgA is distinct from chicken IgG, and IgM. 4. Chicken IgA does not show antigenic homology to human IgA. 5. SDS poly-acrylamide gel electrophoresis revealed IgA to possess heavy chains of 60,000 and light chains of 24,000 mol. wt, respectively. 6. Peptide mapping of tryptic digests of chicken alpha chains reveals approximately 35 peptides. The peptide map pattern is distinct from chicken gamma chains.     

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.     

Investigation of immunological relationships among myosin light chains and troponin C.

Two classes of myosin light chains can be distinguished functionally: those that restore calcium regulation to "desensitized" scallop myofibrils, and those that do not (Kendrick-Jones, J., et al. (1976), J. Mol. Biol. 104, 747--775). Despite this functional classification, chemical analyses reveal few patterns unique to regulatory light chains, and, indeed, sequence comparisons suggest structural similarities between both classes of myosin subunits (Collins, J. H. (1977), Nature (London) 259, 699--700; Kendrick-Jones, J., and Jakes, R. (1977), in International Symposium on Myocardial Failure at Tegernsee, Riecker, G., and Boehringer, Ed., Munich, West Germany, Springer-Verlag, pp. 28--40). Immunological assays using antisera to regulatory and to nonregulatory light chains showed no correlation between antigenic activity and the presence or absence of regulatory function. Weak cross-reactivity was observed, however, among myosin light chains and troponin C, consistent with the suggestion made on the basis of sequence homologies that these subunits contain similar structural domains (Weeds, A. G., and McLachlan, A. D. (1974), Nature (London) 252, 646--649). Unexpectedly, the strongest cross-reactivity observed was that between the vertebrate myosin alkali 1 and DTNB light chains.     

Physicochemical and immunological properties of myosin light chains from the ordinary muscle of marine teleost fishes

1. Myosin light chains (A1, A2 and DTNB light chain) were isolated from the ordinary muscle of six marine fishes and the physicochemical and immunological properties were examined. 2. All the isolated light chains were rich in aspartic and glutamic acids, alanine and lysine, but poor in histidine and tyrosine. The contents of alanine, proline and lysine were generally higher in A1 than in A2. Between closely related species such as skipjack and bonito, the amino acid profiles of corresponding light chains were very similar to each other. 3. Structural similarity and difference of A1 light chain were further demonstrated immunologically using anti-A1 antiserum. Precipitation lines were fused with each other among closely related species, whereas strong spurs were observed among phylogenetically distinct species.     

Isolation and distribution of myosin isoenzymes in chicken pectoralis muscle

The preparation of rabbit antibodies uniquely specific for the alkali 1 (A1) and alkali 2 (A2) light chains of chicken pectoralis myosin has led to the direct isolation of two homodimeric species of myosin: A1-myosin and A2-myosin, molecules which contain the same light chain on each head. The existence of a heterodimeric species, containing both A1 and A2 light chains, was also inferred. The three types of alkali light chain isoenzymes occur in approximately equal amounts in adult chicken pectoralis muscle.The specificities of the antibodies were determined by modified Farr and solid phase radioimmunoassays, as well as by antibody-affinity chromatography. The determinants in myosin that are recognized by the purified antibodies appear to be confined to the N-terminal sequences of the alkali light chains. As a result of this narrow specificity, these immunological reagents can be used to characterize the distribution of A1 and A2 within the myosin molecule, and to localize the individual light chains within the muscle.By labeling the antibodies with a fluorescent marker we have shown that A1 and A2 are present within each myofibril, as well as within the same fiber (Lowey et al., 1979a). Moreover, by using goat anti-rabbit immunoglobulin to enhance the visualization of the primary antibodies against the light chains, we have demonstrated in the electron microscope that A1 and A2 co-exist along the length of each myofilament. This observation suggests that whatever functional differences may exist among the alkali light chain isoenzymes, they must operate within the constraints of a single filament.     

Myosin subunit interactions. Localization of the alkali light chains

Myosin homodimers, molecules containing either the A1 or the A2 light chain, do not exchange their light chains under conditions approximating physiological temperature and ionic strength. Myosin heterodimers, molecules containing both A1 and A2 light chains, are therefore formed at the time of synthesis rather than by a labile subunit exchange. Antibodies specific for the amino-terminal region of the alkali light chains were used to localize these subunits in myosin by immunoelectron microscopy. The close proximity of the alkali light chain to the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain in the "neck" region of the myosin head is consistent with the finding that the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain influences subunit interactions between the alkali light chain and heavy chain in vertebrate skeletal muscle myosin.     

Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.     

Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.     

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.     

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.     

Size and charge requirements for kinetic modulation and actin binding by alkali 1-type myosin essential light chains.

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by 1H NMR that the major actin binding region occurred in the N-terminal four residues, APKK. These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys3 or Lys4 with Ala resulted in increased Km and kcat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased Km and kcat still further. Alteration of Ala1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain's ability to bind actin. Furthermore, we found a significant correlation between the apparent Km for actin and the kcat for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.     

Physico-chemical studies on the light chains of myosin. 3. Evidence for a regulatory role of a rabbit myosin light chain

Treatment of myosin with DTNB causes a decrease in the calcium sensitivity of actomyosin, concurrently with the release of the DTNB light chains. The removal of the calcium-binding DTNB light chains is accompanied by a loss of the calcium binding capacity of myosin. A regulatory role is ascribed to these light chains.     

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.     

Sequencing of laminin B chain cDNAs reveals C-terminal regions of coiled-coil alpha-helix.

cDNAs for laminin B chains have been isolated from a parietal endoderm cDNA library in pUC8 and pUC9. Identification is based on: ability to direct the synthesis in Escherichia coli of polypeptides carrying laminin antigen determinants, in vitro translation of hybrid selected mRNA, and hybridization to high mol. wt. RNA differentially expressed in cells synthesizing large amounts of laminin. The plasmid pPE9 hybrid selects mRNA for the B2 (mol. wt. 185 000) chain and provides 217 residues of C-terminal amino acid sequence. The plasmids pPE386 and 49 both hybrid select mRNAs for the B1a (mol. wt. 205 000) and B1b (mol. wt. 200 000) chains. These two cDNAs are identical over much of their sequence, but pPE386 includes 133 nucleotides of 3' non-coding sequence and a poly(A) tail. Together they provide 495 residues of C-terminal amino acid sequence. Analysis of the predicted sequences reveals a striking heptad repeat, with a high probability that residues a and d are hydrophobic. Such a repeat is typical of the coiled-coil alpha-helices found in proteins such as myosin, tropomyosin and desmin (2-stranded) and fibrinogen (3-stranded).     

Inability of the smallest light chain to bind to fetal fast muscle myosin.

1. The smallest light chain of myosin, g3, was not transferred from adult HMM to fetal myosin in alkali (pH 10.5) under conditions when the light chains dissociated from myosin. 2. The g3 isolated from adult myosin did not bind to fetal myosin at either pH 7.8 or 10.5.