Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Invited Review: plasticity and energetic demands of contraction in skeletal and cardiac muscle.

Numerous studies have explored the energetic properties of skeletal and cardiac muscle fibers. In this mini-review, we specifically explore the interactions between actin and myosin during cross-bridge cycling and provide a conceptual framework for the chemomechanical transduction that drives muscle fiber energetic demands. Because the myosin heavy chain (MHC) is the site of ATP hydrolysis and actin binding, we focus on the mechanical and energetic properties of different MHC isoforms. Based on the conceptual framework that is provided, we discuss possible sites where muscle remodeling may impact the energetic demands of contraction in skeletal and cardiac muscle.     

Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.     

Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.     

Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.     

Adenylate deaminase. A multigene family in humans and rats

Multiple AMP deaminase (AMP-D) isoforms have been found in vertebrates, and tissue-specific inherited deficiencies of AMP-D have been described in two unrelated clinical syndromes suggesting there may be more than one AMP-D gene in higher eukaryotes. Using a newly isolated cDNA cloned from an adult rat brain library and a previously reported cDNA cloned from adult rat skeletal muscle, two linked AMP-D genes have been identified in rat and man. ampd1 is expressed at high levels in skeletal muscle of the adult rat. ampd2 is the predominant gene expressed in non-muscle tissues and smooth muscle of the adult rat, and it is also the predominant gene expressed in embryonic muscle and undifferentiated myoblasts. Both genes are expressed in cardiac muscle of the adult rat. The peptides encoded by these two genes have distinct immunological properties. The conservation of nucleotide sequence and exon/intron boundaries in these two genes suggests they arose by duplication of a common primordial gene around 150 million years ago.     

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.     

Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin

We have previously shown that inhibition of the ATPase activity of skeletal muscle myosin subfragment 1 (S1) by caldesmon is correlated with the inhibition of S1 binding in the presence of ATP or pyrophosphate (Chalovich, J., Cornelius, P., and Benson, C. (1987) J. Biol Chem. 262, 5711-5716). In contrast, Lash et al. (Lash, J., Sellers, J., and Hathaway, D. (1986) J. Biol. Chem. 261, 16155-16160) have shown that the inhibition of ATPase activity of smooth muscle heavy meromyosin (HMM) by caldesmon is correlated with an increase in the binding of HMM to actin in the presence of ATP. We now show, in agreement, that caldesmon does increase the binding of smooth muscle HMM to actin-tropomyosin while decreasing the ATPase activity. The effect of caldesmon on the binding of smooth HMM is reversed by Ca2+-calmodulin. Caldesmon strengthens the binding of smooth S1.ATP and skeletal HMM.ATP to actin-tropomyosin but to a lesser extent than smooth HMM.ATP. Furthermore, this increase in binding of smooth S1.ATP and skeletal HMM.ATP does not parallel the inhibition of ATPase activity. In contrast, in the absence of ATP, all smooth and skeletal myosin subfragments compete with caldesmon for binding to actin. Thus, the effect that caldesmon has on the binding of myosin subfragments to actin-tropomyosin depends on the source of myosin, the type of subfragment, and the nucleotide present. The inhibition of actin-activated ATP hydrolysis by caldesmon, however, is not greatly different for different smooth and skeletal myosin subfragments. Evidence is presented that caldesmon inhibits actin-activated ATP hydrolysis by attenuating the productive interaction between myosin and actin that normally accelerates ATP hydrolysis. The increased binding seen by some myosin subfragments, in the presence of ATP, may be due to binding of these subfragments to a nonproductive site on actin-caldesmon. The subfragments which show an increase in binding in the presence of ATP and caldesmon appear to bind directly to caldesmon as demonstrated by affinity chromatography.     

Differences between smooth and skeletal muscle myosins in their interactions with F-actin

Myosin and F-actin were prepared from bovine carotid arterial smooth muscle and the properties of the binding of myosin to F-actin were compared with those of the binding of skeletal muscle myosin to F-actin. The following differences were observed between skeletal and smooth muscle myosins. 1. The rate of ATP-induced dissociation of arterial actomyosin was equal to that of hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin, but was much lower than those of skeletal muscle actomyosin and of hybrid actomyosin reconstituted from skeletal muscle myosin and arterial F-actin. 2. The amount of ATP necessary for complete dissociation of arterial actomyosin was 2 mol/mol of myosin, although it is well known that skeletal muscle actomyosin is dissociated completely by the addition of 1 mol ATP per mol of myosin. 3. Arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin did not dissociate upon addition of 0.1 mM PPi, while skeletal muscle actomyosin dissociated completely. 4. In the absence of Mg2+, neither dissociation by ATP nor ATPase [EC 3.6.1.3] activity was observed with arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin. On the other hand, skeletal muscle actomyosin dissociated almost completely upon addition of ATP and showed a considerably high ATPase activity. These observations reveal marked differences between myosins from skeletal and smooth muscles in their binding properties to F-actin.     

Transitions of the left poly-L-proline II helix-right alpha helix type in the regions of myosin subfragment S2 of cross-striated muscle as a possible conformational basis for a mechanical-chemical act]

Local conformational states of fibrous fragments of myosin molecules from striated muscle have been studied. Analysis of the amino acid sequences of the rat embryonic skeletal muscle myosin heavy chain and the nematode myosin heavy chain have been performed with the aim to estimate the influence of electrostatic interactions on secondary structure stability of these fragments. The heterogeneity of stability of alpha-helical conformation along the fibrous fragment have been found on the basis of estimation of interaction between side group charges and between side group charges and main chain charges. Periodically located short sections have been found in the N-terminal half of the myosin rod where clusters of Asp and Glu destabilize alpha-helical structure being ionized. Changes of the distribution of charges near the latter sections bring about conformational transitions from left-handed polyproline II helix to right alpha-helix or vice versa. The new scheme of orientation of fibrous part of the cross-bridge in relation to the thick filament for various stages of muscle contraction process suggests asynchronous character of transitions in the different sites. It may be proposed that existence of alteration left- and right-helical fragments in N-terminal half of fibrous part of heavy myosin chain determines zigzag form of this part of myosin molecule in resting muscle. A model of the cross-bridge movement in the course of ATP hydrolysis has been suggested.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

Studies on Ca2+-Mg2+ binding sites of frog skeletal muscle myosin.

From skeletal muscle myosin light chains readily dissociate from the myosin oligomer in the absence of divalent cations, and unlike rabbit skeletal muscle myosin light chains, the released light chains of frog skeletal muscle myosin have a high Ca2+ binding affinity. Whereas each Ca2+ binding light chain of frog skeletal muscle myosin, when in association with the heavy chains bound 1 mol of Ca2+, when in the dissociated state bound 0.5 mol of Ca2+; the latter were readily displaced with low Mg2+ concentrations. Whereas 10(-5) M Mg2+ displaced all of the Ca2+ binding sites on the released light chains at Ca2+ concentration ranges of 10(-7) to 10(-4) M, there was negligible displacement of the Ca2+ binding sites with native frog skeletal muscle myosin under these same conditions.     

Binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin

The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F-actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5-times weaker in the presence of Ca²⁺ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4-times higher in the presence of Ca²⁺.     

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

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-alpha 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-alpha myosin heavy chain from the 21st day of gestation, whereas nuclear bag1 fibres only acquired reactivity to anti-alpha myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag2 fibres to anti-alpha 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.     

Identification of myosin in Nitella flexilis.

A myosin B-like protein was extracted from the alga Nitella flexilis. SDS-polyacrylamide gel electrophoresis revealed the presence of myosin heavy chain and actin as the main components. At high ionic strength, its ATPase [EC 3.6.1.3] reaction was activated by EDTA or Ca2+ and inhibited by Mg2+. At low ionic strength, superprecipitation was induced by the addition of ATP. Myosin was purified from Nitella myosin B. The molecular weight of the heavy chain of Nitella myosin, estimated by SDS-gel electrophoresis, was slightly higher than that of skeletal muscle myosin. At low ionic strength, Nitella myosin aggregated to form bipolar filaments about 0.2 micron long. At high ionic strength, its ATPase reaction was activated by EDTA or Ca2+, and inhibited by Mg2+. The Mg2+-ATPase reaction of Nitella myosin was activated by skeletal muscle F-actin.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

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.