The regulatory proteins of the myofibril. Characterization and properties of the inhibitory factor (troponin B)

1. Gel-filtration results indicate that the major component of inhibitory-factor preparations isolated by dissociation of the troponin complex consisted of a protein of subunit weight 23000 daltons. By the same procedure a molecular weight of 18000 was obtained for the calcium-sensitizing factor. 2. The inhibitory factor is specific for the actomyosin type of ATPase and ITPase. It is effective on desensitized actomyosin, natural actomyosin and intact myofibrils. 3. For inhibition, the actomyosin ATPase must be stimulated by Mg(2+), Ca(2+) or Mn(2+). The Co(2+)-, Cd(2+)- or Zn(2+)-stimulated ATPases are not affected. 4. Biological activity is stable to treatment with dissociating agents, heat, pH11, pH1 and carboxymethylation. 5. Increasing amounts of actin, but not myosin or tropomyosin, progressively neutralize the inhibitory activity when added to desensitized actomyosin or myofibrils.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

The contractile proteins of smooth muscle. Properties and components of a Ca2+-sensitive actomyosin from chicken gizzard.

The preparation and characterization of a Ca²⁺-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg²⁺ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca²⁺ sensitivity were confined to the neutral pH region. The subunit composition of the Ca²⁺-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

Calcium-sensitive modulation of the actomyosin ATPase by fodrin

Fodrin, a spectrin-like protein isolated from brain, is a long flexible molecule which binds calmodulin and cross-links F-actin. The effects of fodrin on the actin-activated ATPase of myosin have been examined. When added after ATP, fodrin inhibited the actomyosin ATPase. Two to three times as much fodrin was required for inhibition in the presence of Ca2+ as in its absence. Complete inhibition in the absence of Ca2+ occurred at about one fodrin to 200 actins. Inhibition does not appear to result from fodrin cross-linking F-actin, and, thereby, preventing the myosin filaments from reaching the actin filaments; but cross-linking may promote inhibition by trapping the myosin filaments within the cross-linked F-actin. When added before ATP, fodrin stimulated the actomyosin ATPase almost 3-fold in the presence of Ca2+ and by less than 50% in the absence of Ca2+. Stimulation is thought to result from fodrin cross-linking F-actin. After several minutes the stimulations in Ca2+ were greatly reduced, and in the absence of Ca2+ the actomyosin ATPases were substantially inhibited. Whether added before or after ATP, fodrin inhibited the actin-activated ATPase of myosin subfragment 1. This inhibition was also slightly Ca2+ sensitive.     

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin-activated Mg2+-ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+-ATPase activity of these preparations was related in a non-linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+-ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin-activated Mg2+-ATPase that was not Ca2+-sensitive, whereas the Mg2+-ATPase of partially phosphorylated myosin preparations was Ca2+-sensitive.     

A solid-liquid biphasic model for characterization of properties of muscle and platelet contractile proteins.

Actomyosin, myosin, and actin from different sources are adsorbed, apparently as a monolayer, by polystyrene particles teins for 1 mg of Lytron were about 10-7 liters mol-1, while heterogeneity indices (alpha) varied from 0.70 to 1.0 presumably as a function of spontaneous aggregation in the liquid phase. Adsorption was irreversible. Orientation of absorbed molecules permitted association of bound muscle actin with platelet or muscle myosin. The association constant of the former reaction was 2.78 times 10-6 liters mol-1. Enzymatic properties of adsorbed actomyosin, Mg2+ATPase activity was abolished, but association of myosin with bound actin, or association of actin with bound myosin was accompanied by restoration of Mg2+ATPase activity. Every subunit of F-actin strands, unless F-actin had been fully depolymerized to G-actin, could bind myosin and activate Mg2+ATPase activity. Immunogenic characteristics of muscle myosin were enhanced by Lytron adsorption. Elicited antibodies showed selective specificity for an antigenic determinant located near or at the actin combining site of muscle myosin. Antibodies did not react with actomyosin. Antibodies prevented association of actin with muscle myosin because they inhibited both superprecipitation and development of Mg2+ATPase activity.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Myosin-linked calcium regulation in vertebrate smooth muscle.

By the use of a new procedure, actomyosin may be extracted in high yield and purity from fowl gizzard which exhibits a calcium-dependent actin-activated ATPase activity comparable to that of the parent myofibril-like preparation. Studies of this vertebrate smooth muscle actomyosin show that the regulation of the actin-myosin interaction is effected, as in molluscan muscles, by the myosin molecule itself and not by an actin-linked regulatory system, as found in vertebrate skeletal muscle.Thus, calcium-sensitive smooth muscle actomyosin is composed of only myosin, actin and tropomyosin, any troponin-like components being absent. Myosin is the only component that binds significant amounts of calcium and shows a calcium-dependent actin-activated ATPase activity in the presence of F-actin from either gizzard or rabbit skeletal muscle.The cross-reaction of gizzard thin filaments with skeletal muscle myosin produces an actomyosin whose actin-activated ATPase is calcium-insensitive, showing that smooth muscle thin filaments do not serve a regulatory function.The effect of Mg²⁺ and pH, and evidence for the involvement of one of the myosin light chains in calcium regulation are described and discussed.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Functional integrity of the SH1 site in myosin from hypertrophied myocardium.

The hypothesis that an alteration in the SH1 site of hypertrophy myosin is reponsible for the reduced Ca2+-stimulated ATPase activity is examined.The functional integrity of the SH1 site was evaluated by measurement of the (K+)-EDTA-stimulated and Mg2+-inhibited ATPase activities. Neither activity differed from control although the Ca2+-stimulated ATPase of the same preparations was significantly reduced. The reduction in Ca2+-activated ATPase was independent of ionic strength. Titration with N-ethylmaleimide elevated the Ca2+-stimulated ATPase of hypertrophy myosin to the same peak activity as control. Actin-stimulated ATPase activity of hypertrophy myosin was also reduced. The results indicate that the SH1 of hypertrophy myosin is functionally intact for (K+)EDTA-stimulated ATPase and Mg2+ inhibition, but functionally deficient with regard to Ca2+-stimulated and actin-activated ATPase activities. This implies a partition of the functional aspects of SH1.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Isolation and localization from chicken gizzard of an inhibitory protein for Mg2+-activated skeletal muscle actomyosin ATPase.

An inhibitory protein for Mg2+-activated actomyosin ATPase from rabbit skeletal muscle was prepared from frozen chicken gizzard and purified by DEAE-Sephadex chromatography and gel filtration. 2. The inhibition by this protein was released by the addition of skeletal muscle troponin C and was independent of gizzard tropomyosin. 3. Localization of the inhibitory protein in gizzard muscle tissue and gizzard thin filaments was demonstrated by immunohistological techniques and immunodiffusion tests.     

Inhibition of actomyosin ATPase by high concentrations of 5-hydroxytryptamine. Possible basis of lesion in 5HT-induced experimental myopathy.

The effect of 5-hydroxytryptamine (5HT) on the ATPase activity and sulphydryl group reactivity of mammalian skeletal muscle actomyosin has been studied. 5HT inhibited the Mg2+-activated but not the Ca2+-activated ATPase activity of actomyosin. It slightly activated myosin ATPase. The sulphydryl groups of actomyosin reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) were blocked by concentrations of 5HT which inhibited the Mg2+-activated ATPase. The significance of the results are discussed in relation to the muscle lesions in the experimental myopathy induced by 5HT and imipramine.     

Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon

Caldesmon, a major calmodulin- and actin-binding protein of smooth muscle (Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5652-5655), has been obtained in highly purified form from chicken gizzard by a modification of a previously published procedure (Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870) and was found to cause a significant inhibition of both superprecipitation and actin-activated myosin Mg2+-ATPase activity in a system reconstituted from the purified contractile and regulatory proteins without influencing the phosphorylation state of myosin. This inhibitory effect was seen both in the presence and absence of tropomyosin. A Ca2+-and calmodulin-dependent kinase which catalyzed phosphorylation of caldesmon was identified in chicken gizzard; this kinase is distinct from myosin light-chain kinase. Caldesmon prepared by calmodulin-Sepharose affinity chromatography was contaminated with caldesmon kinase activity and was unable to inhibit actomyosin ATPase activity or superprecipitation. Phosphatase activity capable of dephosphorylating caldesmon was also identified in smooth muscle. These results indicate that caldesmon can inhibit smooth muscle actomyosin ATPase activity in vitro, and this function may itself be subject to regulation by reversible phosphorylation of caldesmon.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.