The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin

The dependence of the onset and course of turbidity changes ( superprecipitation) induced by ATP were studied in a natural actomyosin suspension with the dephosphorylated and phosphorylated forms of light chains (LC2) of myosin. It was found that the onset and time course of the changes in turbidity of the natural actomyosin suspension are strongly dependent on the (phosphorylated and dephosphorylated) form of these chains of myosin. The ATPase activity of actomyosin with phosphorylated LC2 was lower and the half-time for achieving maximal turbidity of actomyosin suspension after addition of ATP was higher than that of actomyosin with dephosphorylated LC2. Natural actomyosin preparations contain endogenous light-chain kinase and phosphatase. The changes of turbidity induced by ATP in the natural actomyosin suspension are greatly diminished in the presence of phosphate. Thiophosphorylation of LC2 of myosin leads to a decrease of the extent of superprecipitation of natural actomyosin. The release of [32P]phosphate from actomyosin containing [32P]ATP-phosphorylated LC2 of myosin increases with increased turbidity of actomyosin suspension. The change of the form LC2 as a kind of additional myosin-linked regulation of superprecipitation is discussed.     

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.     

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.     

The Precursors Affecting the Composition of Capsaicin and Its Analogues in the Fruits of Capsicum annuum var. annuum cv. Karayatsubusa

The effects of tropomyosin and troponin on the heat-induced gelation of myosin were investigated by SDS-polyacrylamide gel electrophoresis, scanning electron microscopy and gel rigidity assay, in comparisons with natural and desensitized actomyosin. SDS-polyacrylamide gel electrophoretograms revealed that tropomyosin was almost completely removed from each desensitized actomyosin samples while it was retained in natural actomyosin samples. In spite of this, no significant differences were found in rigidity between natural and desensitized actomyosin gels. No differences could be observed in the microstructure of either actomyosin gel. It may, therefore, be concluded that tropomyosin does not affect the gel texture of the actomyosin system.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.     

Influence of hyperthyroidism on the superprecipitation response and Ca-sensitivity of natural actomyosin in cardiac and skeletal muscle

In cardiac natural actomyosin prepared from hyperthyroid rabbits, the time of onset of the superprecipitation response was shortened by 58% and the rate of response was increased 4-fold compared with euthyroid animals. However, Ca²⁺-sensitivity of cardiac natural actomyosin prepared from either euthyroid or hyperthyroid rabbits was not changed over the range of 10⁻⁷ to 6.6·10⁻⁵ mM free Ca²⁺ concentrations. Skeletal natural actomyosin prepared from either euthyroid or hyperthyroid rabbits showed a far higher Ca²⁺-sensitivity than cardiac natural actomyosin, but there was no difference in either time of onset or rate of superprecipitation response.     

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.     

Influence of variuos ultrasound regimens on oxide-modified actomyosin superprecipitation reaction from skeletal muscle of rabbit

A comparative study of rabbit skeletal muscles oxide-modified actomyosin superprecipitation reaction in dependence on continuous and impulsive (2 ms) ultrasound regimens was studied. From the analyses of kinetic curves the effect of the value of superprecipitation (A(m) - A(0)), time t1/2, required for achievement of half of its value was determined, and the normalized maximal rate of this reaction V was also calculated. It is shown that the use of continuous ultrasound to oxide-modified actomyosin was associated with a significant decrease of superprecipitation relative to controls. However, pulsed ultrasound caused a significant increase in superprecipitation value except for the values (A(m) - A(0)) in the application of the intensity of 0.2 W/cm2. The oxide-modified actomyosin superprecipitation value under the effect of continuous and impulsive ultrasound at intensity 1 W/cm2 in relative to control and all other applied intensities decrease to the most extent. It is caused perhaps by thermal influence of ultrasound. In general, the data obtained give reason to assume that the effects of continuous and pulsed ultrasound on the reaction of oxide-modified proteins complex superpretsipitatsii identical.     

Cardioprotection through a PKC-dependent decrease in myofilament ATPase

Activation of myocardial kappa-opioid receptor-protein kinase C (PKC) pathways may improve postischemic contractile function through a myofilament reduction in ATP utilization. To test this, we first examined the effects of PKC inhibitors on kappa-opioid receptor-dependent cardioprotection. The kappa-opioid receptor agonist U50,488H (U50) increased postischemic left ventricular developed pressure and reduced postischemic end-diastolic pressure compared with controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine whether kappa-opioid-PKC-dependent decreases in Ca2+-dependent actomyosin Mg2+-ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase-lowering protocols. We observed that moderate decreases in myofibrillar ATPase were equally cardioprotective as kappa-opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a kappa-opioid-induced increase in myofilament-associated PKC-epsilon, and myofibrillar Ca2+-independent PKC activity was increased after kappa-opioid stimulation. This PKC-myofilament association led to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC-epsilon activation and translocation to the myofilaments causes a decrease in actomyosin ATPase, which contributes to the kappa-opioid receptor-dependent cardioprotective mechanism.     

DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.     

Microtubules mediate changes in membrane cortical elasticity during contractile activation

The mechanical properties of living cells are highly regulated by remodeling dynamics of the cytoarchitecture, and are linked to a wide variety of physiological and pathological processes. Microtubules (MT) and actomyosin contractility are both involved in regulating focal adhesion (FA) size and cortical elasticity in living cells. Although several studies have examined the effects of MT depolymerization or actomyosin activation on biological processes, very few have investigated the influence of both on the mechanical properties, FA assembly, and spreading of fibroblast cells. Here, we examine how activation of both processes modulates cortical elasticity as a function of time. Enhancement of contractility (calyculin A treatment) or the depolymerization of MTs (nocodazole treatment) individually caused a time-dependent increase in FA size, decrease in cell height and an increase in cortical elasticity. Surprisingly, sequentially stimulating both processes led to a decrease in cortical elasticity, loss of intact FAs and a concomitant increase in cell height. Our results demonstrate that loss of MTs disables the ability of fibroblast cells to maintain increased contractility and cortical elasticity upon activation of myosin-II. We speculate that in the absence of an intact MT network, a large amount of contractile tension is transmitted directly to FA sites resulting in their disassembly. This implies that tension-mediated FA growth may have an upper bound, beyond which disassembly takes place. The interplay between cytoskeletal remodeling and actomyosin contractility modulates FA size and cell height, leading to dynamic time-dependent changes in the cortical elasticity of fibroblast cells.     

Structural studies of natural actomyosin from thermally acclimated frogs.

Natural actomyosin was isolated from skeletal muscle of frogs (Rana catesbeiana) acclimated at 25 degrees C and 5 degrees C. It was found that preparations isolated from warm-acclimated frogs may display considerable degradation of myosin heavy chains as compared with preparations isolated from cold-acclimated frogs. However, degradation may be minimized by inclusion of protease inhibitors during purification, indicating enhanced protease activity in preparations of natural actomyosin from warm-acclimated frogs. When purified in the presence of protease inhibitors, natural actomyosin from both warm-acclimated and cold-acclimated frogs exhibits comparable subunit composition of SDS-gel electrophoresis. The overall gel pattern is similar to that obtained from rabbit natural actomyosin except that in the frog, troponin-T and troponin-C appear to co-migrate with tropomyosin and myosin light chain 2, respectively.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

Changes in the mechano-chemical properties of actomyosin during desensitization

The loss of Ca2+-sensitivity by natural actomyosin (desensitisation) after treatment with low ionic strength solutions results in marked deceleration of protein superprecipitation. This phenomenon is not due to the removal of minor proteins, since a similar effect was observed during "desensitisation" of synthetic actomyosin containing only myosin and actin. However, addition to desensitised actomyosin of tropomyosin, especially in combination with alpha-actinin markedly restores the initial parameters of superprecipitation and ATPase activity. It was assumed that desensitisation has a direct modifying influence on actomyosin, whose effect is weakened in the presence of tropomyosin and alpha-actinin.     

Some effects of pressure treatment on actomyosin systems.

Natural actomyosin, actin and myosin, have been pressurized at up to 150 MN/m2 for 1 h at 0 degrees C and examined 3-5 h later. Pressurization of myosin resulted in the formation of aggregates with a molecular weight approximately that expected for a dimer, whereas with F-actin depolymerization occurred. With actomyosin, a gel to sol transition was promoted. Viscosity and light-scattering measurements indicated that pressurization results in a large measure of disaggregation of actomyosin in solution. Pressurization of actomyosin resulted in a greater decrease in the calcium-sensitive, than in the calcium-independent, Mg2+ ATPase activity. The Ca2+ and K+-EDTA ATPase activities of myosin were inhibited to about the same extent.     

The inhibitory action of the light meromyosin component on the myofibrillar and actomyosin atp-ase.

The protein component of light meromyosin [LMM-1] was shown earlier to relax glycerinated muscle fibres and actomyosin. Presently its influence on ATP-ase activity of myofibrils, actomyosin, myosin and heavy meromyosin has been studied. LMM-1 decreases Mg-ATP-ase activity of myofibrils and of reconstructed actomyosin by 25-- 30% and does not change [or slightly increases] Ca-ATP-ase activity of this protein and of myosin; besides LMM-1 is able to increase Mg-ATP-ase of HMM substantially. LMM-1 markedly inhibits [preliminary data] the activation of ATP-ase activity of HMM by actin. It is suggested that LMM-1 protein interacts with myosin and decreases the actin-myosin affinity, displacing actin out of the complex. It reacts only with one of the heads of myosin. Probably this suggestion can account for a relatively slight inhibition of ATP-ase activity of complex by LMM-1. LMM-1 represents a natural and specific inhibitor of Mg-AM-ATP-ase activity, included in the structure of myosin protofibrils and interacting with the myosin active site region.     

Phosphorylation of guinea pig cardiac natural actomyosin and its effect on ATPase activity.

Guinea pig cardiac natural actomyosin incubated with commercial protein kinase, Mg²⁺-ATP, and cyclic AMP produced little or no change in actomyosin ATPase activity. However, addition of sodium fluoride, a known phosphatase inhibitor resulted in a decreased actomyosin ATPase at all measured calcium concentrations. The presence of phosphatase activity in actomyosin and protein kinase was confirmed with ?-nitrophenyl phosphate. These results indicate the importance of inhibiting phosphatase activity, particularly when measuring biological or enzymatic activity as a function of phosphorylation.     

Effect of zinc salts on the structure–function of actomyosin from pelagic fish

The effect of zinc salts, zinc chloride and zinc sulfate on the structure and ATPase enzyme activity of actomyosin from pelagic fish (Sardinella longiceps) has been investigated. ATPase enzyme activity decreased in the presence of both the zinc salts. The inhibitory effect is present in both pH of 7.0 and 9.0. At concentration of 1 × 10^?3 M of zinc salts, complete inhibition of ATPase enzyme activity is observed. With the increase in temperature from 25 °C to 45 °C the ATPase activity decreased by nearly 80%. The solubility profile of actomyosin in the presence of zinc salts shows a sigmoid pattern as the concentration of both the zinc salts increases. Free SH content of actomyosin decreased with the increase in concentration of zinc salts. Intrinsic fluorescence indicated significant decrease in relative fluorescence intensity of actomyosin. This indicates significant alterations in the structure of actomyosin. Analysis of secondary structure also indicates significant alteration in the α-helical content upon binding of both zinc salts.     

Effect of caldesmon on the position and myosin-induced movement of smooth muscle tropomyosin bound to actin

It is known that the actin-binding protein caldesmon inhibits actomyosin ATPase activity and might in this way take part in the thin filament regulation of smooth muscle contraction. Although the molecular mechanism of this inhibition is unknown, it is clear that the presence of actin-bound tropomyosin is necessary for full inhibition. Recent evidence also suggests that the myosin-induced movement of tropomyosin plays a key role in regulation. In this work, fluorescence studies provide evidence to show that caldesmon interacts with and alters the position of tropomyosin in a reconstituted actin thin filament and thereby limits the ability of myosin heads to move tropomyosin. Caldesmon interacts with the Cys-190 region in the COOH-terminal half of tropomyosin, resulting in the movement of this part of tropomyosin to a new position on actin. Additionally, this constrains the myosin-induced movement of this region of tropomyosin. On the other hand, caldesmon does not appear to interact with the Cys-36 region in the NH2-terminal half of tropomyosin and neither alters the position of nor significantly constrains the myosin-induced movement of this part of tropomyosin. The ability of caldesmon to limit the myosin-induced movement of tropomyosin provides a possible molecular basis for the inhibitory function of caldesmon. The different movements of the two halves of tropomyosin indicate that actin-bound tropomyosin moves as a flexible molecule and not as a rigid rod. Interestingly, caldesmon, which inhibits tropomyosin's potentiation of actomyosin ATPase activity, moves tropomyosin in one direction, whereas myosin heads, which enhance potentiation, move tropomyosin in the opposite direction.     

Kontraktionseigenschaften von isoliertem Schleimpilzactomyosin

Summary The isolated contractile proteins of the slime mouldPhysarum polycephalum and of rabbit skeletal muscle were investigated by using actomyosin thread models. The actomyosins were compared with respect to contraction behaviour, fine structure, and ATPase activity. Thread models were made of natural and synthetic actomyosins of both systems.The natural actomyosins differ considerably: The actin filament length ofPhysarum actomyosin is only about one fourth, the ATPase activity and actin/myosin ratio are much lower compared to natural muscle actomyosin. The contraction rate of the natural slime mould actomyosin is remarkably slower than that of the natural muscle actomyosin.The synthetic actomyosins were formed from separately isolated actins and myosins with a constant actin/myosin ratio and comparable actin filament lengths. The thread models of either recombined and hybridized actomyosins of both systems contract with nearly identical rates. The comparison of the synthetic actomyosins shows that under comparable conditions a) the actomyosins of both systems perform work with the same efficiency, b) the actin and myosin component is freely exchangeable without any change in the rate of actomyosin contraction. These results indicate that in both skeletal muscle and slime mould the force generation is based on the same mechanism of actin-myosin interaction.

---

---

Ein Teil dieser Ergebnisse wurde als Symposiumsvortrag auf dem 9th Meeting of the Federation of the European Biochemical Societies, Budapest vorgetragen.