The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca²⁺-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca²⁺ in the medium. 4. The Ca²⁺-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca²⁺-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.     

Dissociation of actomyosin complex by monovalent fragments of polyclonal antibodies to myosin

The monovalent fragments of antibodies specific for skeletal muscle myosin inhibit myosin ATPase activity and dissociate the actomyosin complex, as shown by analytical ultracentrifugation and viscosity measurements.     

A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized-actomyosin

1. The preparation and properties of a myofibrillar protein factor which inhibits the Mg(2+)-activated adenosine triphosphatase of desensitized actomyosin is described. 2. This factor had negligible effect on the Mg(2+)-activated adenosine triphosphatase of natural actomyosin and on the Ca(2+)-activated adenosine triphosphatases of desensitized actomyosin and myosin. 3. The Mg(2+)-activated inosine triphosphatase activity of desensitized actomyosin was not affected by the factor. 4. The inhibitory effect was sensitive to ionic strength. In addition to their ionic effects Mg(2+) and Ca(2+) appeared to have a specific action in reducing the effect of the inhibitor. 5. F-actin reduced the inhibition whereas Bailey-type tropo-myosin had little effect. 6. As far as can be judged from the reported experiments this factor is different from any of the previously described myofibrillar components.     

The Influence of Hydrogen Ion Concentration on Calcium Binding and Release by Skeletal Muscle Sarcoplasmic Reticulum

Calcium release and binding produced by alterations in pH were investigated in isolated sarcoplasmic reticulum (SR) from skeletal muscle. When the pH was abruptly increased from 6.46 to 7.82, after calcium loading for 30 sec, 80–90 nanomoles (nmole) of calcium/mg protein were released. When the pH was abruptly decreased from 7.56 to 6.46, after calcium loading for 30 sec, 25–30 nmole of calcium/mg protein were rebound. The calcium release process was shown to be a function of pH change: 57 nmole of calcium were released per 1 pH unit change per mg protein. The amount of adenosine triphosphate (ATP) bound to the SR was not altered by the pH changes. The release phenomenon was not due to alteration of ATP concentration by the increased pH. Native actomyosin was combined with SR in order to study the effectiveness of calcium release from the SR by pH change in inducing super-precipitation of actomyosin. It was found that SR, in an amount high enough to inhibit superprecipitation at pH 6.5, did not prevent the process when the pH was suddenly increased to 7.3, indicating that the affinity of SR for calcium depends specifically on pH. These data suggest the possible participation of hydrogen ion concentration in excitation-contraction coupling.     

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.     

Suppression of superprecipitation of contractile proteins from chicken gizzard muscle by preincubation in the presence of adenosine triphosphate without Ca2+

Superprecipitation of reconstituted actomyosin composed of smooth muscle myosin, skeletal muscle actin and smooth muscle native tropomyosin was studied. When the actomyosin solution was preincubated in the presence of ATP and the absence of Ca2+, or in the relaxed state, superprecipitation was markedly suppressed. The extent of suppression was correlated with the inhibition of the phosphorylation of the 20,000-dalton light chain of smooth muscle myosin. This is consistent with the theory that the interaction of smooth muscle actomyosin is regulated by the phosphorylation of myosin light chain through a system of myosin light chain kinase and phosphatase. However, further studies showed that the myosin light chain kinase and phosphatase system could not explain the present suppression of superprecipitation, even if a cyclic AMP-dependent protein kinase system was also involved. A new regulatory factor should be taken into account in the regulation of smooth muscle actomyosin interaction.     

Role of myocardium and endothelium in coronary vascular smooth muscle responses to hypoxia

Hypoxia triggers a mechanism that induces vasodilation in the whole heart but not necessarily in isolated coronary arteries. We therefore studied the role of cardiomyocytes (CM), smooth muscle cells (SMC), and endothelial cells (EC) in coronary responses to hypoxia (PO(2) of 5-10 mmHg). In an attempt to determine the factor(s) released in response to hypoxia, we inhibited the contribution of adenosine, ATP-sensitive K(+) channels, prostaglandins, and nitric oxide. Isolated rat septal artery segments without (-T) and with a layer of cardiac tissue (+T) were mounted in a double wire myograph, and constriction was induced. Hypoxia induced a decrease in isometric force of 21% and 61% in -T and +T segments, respectively (P < 0.05). EC removal increased the relaxation to hypoxia in -T segments to 33% but had the same effect in +T segments (61%). Only one of the inhibitors, the adenosine antagonist in +T segments, partially affected the relaxation due to hypoxia. The role of adenosine is thus limited and other mechanisms have to contribute. We conclude that hypoxia induces a relaxation of SMC that is augmented by the presence of CM and blunted by the endothelium. A single mediator does not induce those effects.     

Human platelet actin. Evidence of beta and gamma forms and similarity of properties with sarcomeric actin.

Human blood platelet actin was purified using 30% sucrose to extract actomyosin and potassium iodide to dissociate actomyosin and to depolymerize actin. Pure actin thus obtained resembles skeletic muscle actin in its polymerization properties, CD spectra and ability to activate myosin myosin Mg2+-ATPase. Isoelectric focusing gel analysis shows that human blood platelet actin exists in beta and gamma forms. The ratio of beta to gamma forms is of 5 in purified actin, in whole cell extract and in all the fractions studied.     

A model of muscle contraction based on the Langevin equation with actomyosin potentials

We propose a muscle contraction model that is essentially a model of the motion of myosin motors as described by a Langevin equation. This model involves one-dimensional numerical calculations wherein the total force is the sum of a viscous force proportional to the myosin head velocity, a white Gaussian noise produced by random forces and other potential forces originating from the actomyosin structure and intra-molecular charges. We calculate the velocity of a single myosin on an actin filament to be 4.9–49 μm/s, depending on the viscosity between the actomyosin molecules. A myosin filament with a hundred myosin heads is used to simulate the contractions of a half-sarcomere within the skeletal muscle. The force response due to a quick release in the isometric contraction is simulated using a process wherein crossbridges are changed forcibly from one state to another. In contrast, the force response to a quick stretch is simulated using purely mechanical characteristics. We simulate the force–velocity relation and energy efficiency in the isotonic contraction and adenosine triphosphate consumption. The simulation results are in good agreement with the experimental results. We show that the Langevin equation for the actomyosin potentials can be modified statistically to become an existing muscle model that uses Maxwell elements.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of 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.     

Calcium ion-dependent myosin from decapod-crustacean muscles.

Ca2+ regulation of arthropod actomyosin adenosine triphosphatase is associated with both the thin filaments, as in vertebrates, and with the myosin, as in molluscs. The actomyosin of decapod-crustacean fast muscles was previously considered to be an exception, displaying only a Ca2+-regulatory system linked to the thin filaments and not a myosin-linked regulatory system. In the present study, myosin regulation is demonstrated in a variety of decapod muscles when they are tested under more physiological ionic conditions. Myosin regulation is shown by using mixtures of pure rabbit actin with myofibrils, with actomyosin and with purified myosin, and in each case the adenosine triphosphatase is Ca2+ dependent. Myosin regulation may also occur in vertebrate striated muscle, but seemingly is lost during purification of the myosin.     

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris.

Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase.     

The adenosine-triphosphatase activity of desensitized actomysin

1. A simple procedure involving repeated washings of actomyosin, extracted as the complex from myofibrils (natural actomyosin) at ionic strength less than 0.002, is described for the preparation of a desensitized actomyosin. 2. The Mg(2+)-activated adenosine triphosphatase of natural actomyosin was markedly inhibited by ethylenedioxybis(ethyleneamino)tetra-acetic acid, whereas that of the desensitized actomyosin was unaffected. 3. The activity of the Ca(2+)-activated adenosine triphosphatase of natural actomyosin was generally lower than that of the Mg(2+)-activated adenosine triphosphatase, whereas in the desensitized actomyosin the difference between the activities was considerably less. In both natural and desensitized actomyosin the adenosine-triphosphatase activities in the presence of Mg(2+) were similar. 4. The conversion of the natural into the desensitized actomyosin was accompanied by the removal of a protein fraction containing the factors responsible for the sensitivity to ethylenedioxybis(ethyleneamino)tetra-acetic acid and for modifying the Ca(2+)-activated adenosine triphosphatase. When added to a desensitized actomyosin this fraction effected a reversal to the natural form. The recombination was facilitated by increasing the ionic strength of the medium. The two factors showed different stabilities to heat and tryptic digestion.     

Troponin I: Inhibitor or facilitator

TN-I occurs as a homologous group of proteins which form part of the regulatory system of vertebrate and invertebrate striated muscle. These proteins are present in vertebrate muscle as isoforms, Mr 21000-24000, that are specific for the muscle type and under individual genetic control. TN-I occupies a central position in the chain of events starting with the binding of calcium to troponin C and ending with activation of the Ca2+ stimulated MgATPase of the actomyosin filament in muscle. The ability of TN-I to inhibit the MgATPase of actomyosin in a manner that is accentuated by tropomyosin is fundamental to its role but the molecular mechanism involved is not yet completely understood. For the actomyosin ATPase to be regulated the interaction of TN-I with actin, TN-C and TN-T must undergo changes as the calcium concentration in the muscle cell rises, which result in the loss of its inhibitory activity. A variety of techniques have enabled the sites of interaction to be defined in terms of regions of the polypeptide chain that must be intact to preserve the biological properties of TN-I. There is also evidence for conformational changes that occur when the complex with TN-C binds calcium. Nevertheless a detailed high resolution structure of the troponin complex and its relation to actin/tropomyosin is not yet available. TN-I induces changes in those proteins with which it interacts, that are essential for their function. In the special case of cardiac TN-I its effect on the calcium binding properties of TN-C is modulated by phosphorylation. It has yet to be determined whether TN-I acts directly as an inhibitor or indirectly by interacting with associated proteins to facilitate their role in the regulatory system.     

The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin

1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca²⁺-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100° and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca²⁺-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca²⁺-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca²⁺-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.     

The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit.

1. A series of defined peptides which span the complete sequence were produced from troponin I isolated from white skeletal muscle of the rabbit. 2. Two peptides, CF1 (residues 64-133) and CN4 (residues 96-117) inhibited the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition was potentiated by tropomyosin and the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition, unlike that of troponin I and peptides derived from it, was not potentiated by tropomyosin. 4. The most active inhibitor, peptide CN4, was 45-75% as effective as troponin I when compared on a molar basis. The inhibitory peptide, CN4, and also whole troponin I were shown by affinity chromatography to interact specifically with actin. 5. A strong interaction with troponin C was demonstrated with peptide CF2 (residues 1-47), from the N-terminal region of troponin I. Somewhat weaker interactions were shown with peptides CN5 (residues 1-21) and with the inhibitory peptide CN4. 6. The significance of these interactions for the mechanisms of action of troponin I is discussed.