Characterization and quantitation of myosin heavy-chain mRNA from embryonic cardiac tissue

The messenger RNA (mRNA) coding for myosin heavy chain from the 16-day-old chick embryonic cardiac tissue was purified by a rapid isolation procedure and characterized. The mRNA can be translated with fidelity under optimally chosen conditions. The protein synthesized in response to the RNA was a polypeptide of 200,000 molecular weight, identical to the authentic myosin heavy chain from the homologous chick heart tissue. The purity of the mRNA was assessed by electrophoresis in denaturing gels, by immunoprecipitation of the translation product, and by analysis of the kinetics of hybridization with the complementary DNA (cDNA). The cDNA reassociated with myosin heavy-chain mRNA with kinetics characteristic of a pure mRNA. The sequence complexity data indicated that in the 16-day-old chick embryonic heart cells there is a single mRNA sequence coding for myosin heavy chain in contrast to two different mRNA sequences reportedly present in the skeletal muscle cells (M. Patrinou-Georgoulas and H. A. John, 1977, Cell12, 491).     

Thyroid hormone stimulates synthesis of a cardiac myosin isozyme. Comparison of the two-two-dimensional electrophoretic patterns of the cyanogen bromide peptides of cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits.

The CNBr peptides of [14C]carboxymethylated cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits have been compared using a two-dimensional electrophoretic system. The results indicated that there were extensive differences in the peptide "maps" of these heavy chains, which included differences in the distribution of radiolabeled thiol peptides. Also, the patterns of heavy chain peptides from the cardiac myosins have been compared with those produced by the heavy chain myosin isozymes from skeletal muscles. Peptide maps of heavy chains from red skeletal muscle myosin closely resembled the pattern of peptides found with cardiac myosin heavy chains from euthyroid rabbits. However, peptide maps of heavy chains from white skeletal muscle myosin were dissimilar to those of the cardiac myosin isozymes. We conclude that thyroxine administration stimulates the synthesis of a cardiac myosin isozyme with a heavy chain primary structure which is different from either of the skeletal muscle myosin isozymes.     

Serial deletion constructs of human cardiac myosin heavy chain genes generated by PCR amplification

Summary Serial deletion constructs derived from the 5-flanking regions of the human cardiac - and -myosin heavy chain genes were generated by polymerase chain reaction (PCR) amplifications. Generation of different length chimeric constructs were based on the complete sequence of the human cardiac myosin heavy chain genes [1, 2]. The primers were synthesized with HindIII and BamH₁ sites and were linked to any designed nucleotide of the 5 flanking sequence of the myosin heavy chain gene(s). Following the PCR amplification and the site-directed mutagenesis, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenical acetyltransferase (pBLCAT₃) reporter gene which was restricted with Hind III and BamH₁. Neonatal rat cardiocytes were used to assay the promotor activity (i.e. CAT activity) of different lengths of the chimeric constructs of the gene.     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

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.     

Cytoplasmic myosin from Drosophila melanogaster

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.     

Hamy3, a novel type 100 kDa myosin from sunflower

Hamy3, a novel type myosin heavy chain from sunflower is the smallest myosin described so far, with only 900 amino acid residues. One interesting finding in Hamy3 is the glycine to glutamine alteration at residue 741, which corresponds to chicken skeletal muscle myosin glycine 699 (G699). G699 is found in 125 out of 129 myosin sequences and is interpreted in terms of its role as a pivot point for motion in the myosin "lever arm hypothesis". Changes in this crucial part of myosin might indicate a role that is different from the generation of intracellular motility.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii

A low-molecular-weight myosin has been purified 1500-fold from extracts of Dictyostelium discoideum, based on the increase in K+,EDTA-ATPase specific activity. The purified enzyme resembles the single-headed, low-molecular-weight myosins IA and IB from Acanthamoeba castellanii, and differs from the conventional two-headed, high-molecular-weight myosin previously isolated from Dictyostelium, in several ways. It has higher K+,EDTA-ATPase activity than Ca2+-ATPase activity; it has a native molecular mass of about 150,000 and a single heavy chain of about 117,000; the 117,000-dalton heavy chain is phosphorylated by Acanthamoeba myosin I heavy chain kinase; phosphorylation of its heavy chain enhances its actin-activated Mg2+-ATPase activity; and the 117,000-dalton heavy chain reacts with antibodies raised against the heavy chain of Acanthamoeba myosin IA. None of these properties is shared by the low-molecular-weight active fragment that can be produced by chymotryptic digestion of conventional Dictyostelium myosin. We conclude that Dictyostelium contains an enzyme of the myosin I type previously isolated only from Acanthamoeba.     

Absence of phosphates from the myosin heavy chains of striated muscles

This work aimed to determine whether the heavy chains of myosin from different striated muscle were phosphorylated. Myosin and its heavy chains were prepared from cardiac and skeletal muscles of rats injected in vivo with radioactive phosphates.The results for radioactive phosphate localization indicate the absence of phosphate from pure heavy chains and from any of their purified fragments, whatever the striated muscle used. In addition, phosphates are present in the myosin phosphorylated light chain and in a contaminating protein closely associated to the myosin heavy chain.     

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.     

Probing myosin head structure with monoclonal antibodies

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

Two different preparations of subfragment-1 from scallop adductor myosin

Chymotryptic digestion of scallop myosin yielded two different preparations of subfragment-1, having the following features. The major product from chymotryptic digestion of scallop myosin was subfragment-1 (S1) either in Ca-medium or in EDTA-medium. However, the S1 preparations obtained from the digestion in Ca-medium, abbreviated as Ca-S1(CT), had both types of light chain subunits (regulatory light chains (R-LC) and essential light chains (SH-LC], and 100 Kdaltons (Kd) heavy chain subfragments (HCs), whereas the S1 preparations obtained from the digestion in EDTA-medium, ED-S1(CT), had no R-LC, partially fragmented SH-LC (SH-LC), and 90 Kd HCs. On the other hand, Ca-S1(CT) and ED-S1(CT) were practically identical with each other in ATPase activity and in actin-binding ability. The two S1 preparations were also identical in that the Mg-ATPase activity of both S1 and acto-S1 was insensitive to calcium ions. Ca-S1(CT), which contained both R-LC and SH-LC in a stoichiometric amount, was further digested with trypsin, which is known to cleave rabbit skeletal myosin not only at the head-tail junction but also in the head. The tryptic digestion of Ca-S1(CT) appeared, in terms of the SDS-gel electrophoretic pattern, to occur at a much faster rate in Ca-medium than in EDTA-medium, and with a different digestion profile. It is therefore suggested that association of R-LC induces changes in the heavy chain conformation which result in an increase in the proteolytic digestibility of heavy chains and in an alteration of the site of proteolytic cleavage on heavy chains.     

Amino acid sequences of the two kinds of regulatory light chains of adductor smooth muscle myosin from Patinopecten yessoensis

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Identification of recombinant phages containing sequences from different rat myosin heavy chain genes.

The construction and identification of a recombinant plasmid containing a cDNA insert which hybridizes specifically to myosin heavy chain mRNA is described. The plasmid was used as a probe to screen a rat genomic library for recombinant phages containing myosin heavy chain sequences. Six clones with approximately 15 k bp inserts each were isolated. Digestion with several restriction enzymes and hybridization of the fractionated DNA with the plasmid probe showed that the clones contained 3 different DNA inserts. Electron microscopy of a heteroduplex made by hybridization of DNA from two clones confirmed that the inserts originated in different genes. Hybridization of size-fractionated ECOR1 digested rat spleen DNA with the cloned probe suggested the existence of at least 5 myosin heavy chain genes.     

Effects of isometric training on skeletal myosin heavy chain expression

This studytested the hypothesis that an isometric resistance-training programinduces upregulation of slow myosin heavy chain (MHC) expression in afast-twitch skeletal muscle. Thus we studied the effects of tworesistance-training programs on rodent medial gastrocnemius (MG) musclethat were designed to elicit repetitive isometric contractions(10-12 per set; 4 sets per session) of different duration (8 vs. 5 s) and activation frequency (100 vs. 60 Hz) per contraction during eachtraining session (total of 6 and 12 sessions). Results showed that bothtraining paradigms produced significant increases in muscle weight(~11-13%) after completion of training(P < 0.05). Significanttransformations in MHC expression occurred and involved specifically adecrease in the relative expression of the fast type IIb MHC andconcomitant increased expression of the fast type IIx MHC.These adaptations were observed in both the "white" and"red" regions of the MG, and they occurred at both the mRNA andprotein levels. These adaptations were detected after onlysix training sessions. Neither of the training programs produced anychange in the relative expression of either the slow type I MHC or themoderately fast type IIa MHC, which can be upregulated in the red MG bychronic functional overload. These findings show that theisometric protocols used in this investigation were not sufficient toinduce the hypothesized changes in the myosin heavy chain isoformexpression in rodent skeletal muscle.

     

Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres

Summary In the present study we have investigated the reactivity of rat muscle to a specific monoclonal antibody directed against alpha cardiac myosin heavy chain. Serial cross sections of rat hindlimb muscles from the 17th day in utero to adulthood, and after neonatal denervation and de-efferentation, were studied by light microscope immunohistochemistry. Staining with anti- myosin heavy chain was restricted to intrafusal bag fibres in all specimens studied. Nuclear bag₂ fibres were moderately to strongly stained in the intracapsular portion and gradually lost their reactivity towards the ends, whereas nuclear bag₁ fibres were stained for a short distance in each pole. Nuclear bag₂ fibres displayed reactivity to anti- myosin heavy chain from the 21st day of gestation, whereas nuclear bag₁ fibres only acquired reactivity to anti- myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag₂ fibres to anti- myosin heavy chain was decreased and limited to a shorter portion of the fibre, whereas nuclear bag₁ fibres were unreactive. We showed that a myosin heavy chain isoform hitherto unknown for skeletal muscle is specifically expressed in rat nuclear bag fibres. These findings add further complexity to the intricate pattern of isomyosin expression in intrafusal fibres. Furthermore, we show that motor innervation influences the expression of this isomyosin along the length of the fibres.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.