Purification of a multiphosphorylated peptide from canine cardiac myosin heavy chains labeled in tissue culture

Partial acid hydrolysis of canine cardiac myosin heavy chains labeled with [³²P]orthophosphate in myocardial cell culture yielded a peptide having a molecular weight between 700 and 1500 and containing phosphothreonine and phosphoserine. The phosphate-rich peptide of myosin heavy chains produced by partial acid hydrolysis was purified first by Sephadex gel filtration, followed by elution with a gradient of formic acid from a Dowex ion-exchange chromatograph. Further identification of the multiphosphorylated peptide was made using high voltage electrophoresis and amino acid analyses. The data described here demonstrate that partial acid hydrolysis (time dependent) can be used to produce partially acid-stable peptides in a good yield.     

Purification and some properties of the Glu- and Lys-human plasmin heavy chains.

The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the corresponding plasmins. Two major forms of each heavy chain can be eluted, after adsorption to Sepharose/lysine, utilizing a gradient of epsilon-aminocaproic acid as the eluant. The elution profile of these heavy chains is practically identical to the elution behavior previously observed for human Glu- and Lys-plasminogen, and human Glu- and Lys-plasmin adsorbed to these columns. Sedimentation velocity analysis of the heavy chain of human Glu-plasmin, in the presence of epsilon-aminocaproic acid, demonstrated that a gross conformational alteration occurs in this peptide accompanying binding of this amino acid. A much smaller conformational alteration occurs under similar circumstances with the human Lys-plasmin heavy chain. We find that the NH2-terminal peptide released in the Glu-plasminogen to Lys-plasminogen and Glu-plasmin to Lys-plasmin conversions is also released in the Glu-plasmin heavy chain to Lys-plasmin heavy chain conversion. This reaction is catalyzed at a significant rate only by plasmin and not by urokinase. Finally, no strong interaction between streptokinase and the isolated plasmin heavy chains is observed.     

Myosin light chains of guinea-pig striated muscles. Similarities and differences with rat myosin light chains

1. Myosin light chains of guinea-pig striated muscles have been screened by two-dimensional gel electrophoresis and compared to rat myosin light chains. 2. The fast type light chains 1F and 3F, slow type light chains 1S and 2S, and embryonic type light chain 1E are shown to differ in the two rodents; only the fast type light chains 2F co-electrophorese on the gel. 3. In guinea-pig, as in rat, ventricle muscle light chains appear the same as the 1S and 2S light chains and atrial light chain type 1 the same as the 1E light chain. We show that this embryonic light chain of guinea-pig myosin is difficult to identify and may be confused with the adult 1F light chain.     

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.     

Smooth muscle myosin is composed of homodimeric heavy chains.

Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115 microns/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078 microns/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082 microns/s).     

Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii

In previous work from this laboratory, a partially purified protein kinase from the soil amoeba Acanthamoeba castellanii was shown to phosphorylate the heavy chain of the two single-headed Acanthamoeba myosin isoenzymes, myosin IA and IB, resulting in a 10- to 20-fold increase in their actin-activated Mg2+-ATPase activities (Maruta, H., and Korn, E.D. (1977) J. Biol. Chem. 252, 8329-8332). A myosin I heavy chain kinase has now been purified to near homogeneity from Acanthamoeba by chromatography on DE-52 cellulose, phosphocellulose, and Procion red dye, followed by chromatography on histone-Sepharose. Myosin I heavy chain kinase contains a single polypeptide of 107,000 Da by electrophoretic analysis. Molecular sieve chromatography yields a Stokes radius of 4.1 nm, consistent with a molecular weight of 107,000 for a native protein with a frictional ratio of approximately 1.3:1. The kinase catalyzes the incorporation of 0.9 to 1.0 mol of phosphate into the heavy chain of both myosins IA and IB. Phosphoserine has been shown to be the phosphorylated amino acid in myosin IB. The kinase has highest specific activity toward myosin IA and IB, about 3-4 mumol of phosphate incorporated/min/mg (30 degrees C) at concentrations of myosin I that are well below saturating levels. The kinase also phosphorylates histone 2A, isolated smooth muscle light chains, and, to a very small extent, casein, but has no activity toward phosvitin or myosin II, a third Acanthamoeba myosin isoenzyme with a very different structure from myosin IA and IB. Myosin I heavy chain kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or cAMP for activity. The kinase undergoes an apparent autophosphorylation.     

An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical--chemical and immunological characterization of the light chains.

The light chains of chicken breast muscle myosin (alkali 1 and 2, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 1.c.) have been isolated in pure form and characterized with respect to amino acid composition, uv and circular dichroism (CD) spectral properties, and molecular weight. Antibodies specific for each of the light chains have been used to demonstrate the similarity of alkali 1 and 2 (mol wt 21,000 and 16,000, respectively), and the distinctness of these from DTNB 1.c. (mol wt of 18,000). The DTNB 1.c. isolated by a variety of methods were all immunologically identical. Significant cross-reactivity was observed between corresponding rabbit and chicken light chains, confirming other indications of homology between these proteins in the two species. The immunological difference between alkali 1 and 2 was largely accounted for by an N-terminal peptide, rich in proline, alanine, and lysine, which is unique to alkali 1. The presence of antibodies to this peptide in anti-alkali 1 serum suggests an immunological approach to the question of how alkali 1.c. are distributed in myosin.     

Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization

We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

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.     

Myosin heavy chains in fibers of TTX-paralyzed rat soleus and medial gastrocnemius muscles.

The expression of five myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (Sol) and the deep and superficial medial gastrocnemius (dGM, sGM) muscle after 2 and 4 wk of TTX paralysis by using immunohistochemical techniques. In Sol, after 4 wk of paralysis, fibers containing type I MHC were either pure type I (14%) or also contained developmental (D; 76%), IIa (26%), or IIx (18%) MHC. Values for corresponding fibers in dGM were 8.5, 65, 38, and 22%. Also, by 4 wk an increase was seen in the proportions of fibers expressing IIa MHC in Sol (from 16 to 38%) and dGM (from 24 to 74%). In a region of sGM in control muscles containing pure IIb fibers, a major proportion (86%) remained pure after 4 wk of paralysis, with the remainder coexpressing IIb and IIx. The results indicate that TTX-induced muscle paralysis results in an increase in fibers containing multiple MHC isoforms and that the D isoform appears in a major proportion of these hybrid fibers.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

Purification and some properties of rabbit stomach myosin.

Myosin from rabbit stomach was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultracentrifugation and Sepharose 4B chromatography. The myosin composed of one heavy and two light chains as determined by SDS-gel electrophoresis. The molecular weights of the light chains were the same as those of gizzard myosin, about 20,000 and 17,000, respectively. The pH-activity curve and the KCl concentration dependency of Ca-ATPase of the stomach myosin were similar to those of other smooth muscle myosins. The stomach myosin was more resistant to pepsin digestion than skeletal myosin. Other proteolytic enzymes, trypsin, chymotrypsin, papain, and nagarse, digested the myosin in the same way as skeletal myosin.     

Living macrophages phosphorylate the 20,000 dalton light chains and heavy chains of myosin

Brief incubation of rabbit alveolar macrophages in medium containing ³²Pi results in the incorporation of radioactivity into the 20 KD light chains and into the 220 KD heavy chains of myosin. Phosphorylation of the heavy chain is mediated by a kinase that is probably not myosin light chain kinase. Limited proteolysis of the phosphorylated myosin shows that radioactivity is associated with the rod portion of the heavy chain.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

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.