Superprecipitation Of Actomyosin Research Articles

Використання алюмінію та його сполук у біомедичних дослідженнях

Taking into consideration the nanodimension of functional components of living cells, the application of nanotechnology in the biomedical purposes is currently an urgent task. One of these directions is the use of aluminum nanoparticles for molecular diagnostics, targeted delivery of drugs, the development of new pharmaceuticals. The action of aluminum chloride on the regulatory mechanisms of smooth and skeletal muscle contraction and also the effect of aluminum ions on the ATPase activity and superprecipitation of actomyosin of different types of muscles is discussed.

Features of actomyosin superprecipitation reaction in different environment conditions

The influence of divalent cations (Cd 2+ , Zn 2+ , Sr 2+ ) and staphylococcus protein A on the superprecipitation reaction of cardiac muscle actomyosin was investigated using methods of preparative protein chemistry, optical spectroscopy and mechanokinetic analysis. It was shown that the metal ions in the range of concentrations 0.1–5 mM inhibit the Mg 2+ -dependent SPP reaction of actomyosin cardiac muscle and alter the kinetic parameters of this process. It was revealed that protein A modulates superprecipitation dynamics decreasing the maximal value of optical density and time of its half-maximum achievement as well as starting and normalized superprecipitation rates. Thus, investigated factors are able to influence the actin myosin interaction changing their functional parameters of proteins from cardiomyocyte contractile complex. The kinetic characteristics of actomyosin superprecipitation are sensitive to the influence of physicochemical and pharmacological factors and can be used to study their influence on the molecular mechanisms of muscle contraction. Keywords: actomyosin, superprecipitation, kinetic parameters, metal ions, S. aureus protein A, cardiac muscle.

Effect of fullerene C(60) on ATPase activity and superprecipitation of skeletal muscle actomyosin

Creation of new biocompatible nanomaterials, which can exhibit the specific biological effects, is an important complex problem that requires the use of last accomplishments of biotechnology. The effect of pristine water-soluble fullerene C60 on ATPase activity and superprecipitation reaction of rabbit skeletal muscle natural actomyosin has been revealed, namely an increase of actomyosin superprecipitation and Мg2+, Са2+– and K+-ATPase activity by fullerene was investigated. We conclude that this finding offers a real possibility for the regulation of contraction-relaxation of skeletal muscle with fullerene C60.

Effect of multi‐walled iron‐filled carbon nanotubes on ATPase activity and superprecipitation of natural actomyosin

AbstractThe dependence of actomyosin superprecipitation and Ca2+, Mg2+‐ and K+‐ATPase activity inhibition on the concentration of multi‐walled iron‐filled carbon nanotubes (Fe‐MWCNTs) was investigated. For the superprecipitation of actomyosin, a biphasic effect of Fe‐MWCNTs was found. Namely, at low concentrations (10–9 mg/ml) the superprecipitation was activated, while at high concentrations (10–7 and 10–5 mg/ml) its activity was inhibited.

Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells

We have characterized the interaction of syntrophin with F-actin. Subcellular fractionation of cardiac and skeletal muscle tissues showed that alpha-, beta1- and beta2-syntrophins were present in the soluble and the membrane fraction. Syntrophins are known to bind to the dystrophin-glycoprotein complex (DGC), but since the DGC is not present in the soluble fraction, it was concluded that some syntrophin did not associate with the DGC. Native syntrophins purified from the soluble fraction and recombinant syntrophins were both able to bind to F-actin, and binding occurred through several sites on syntrophin, including the second pleckstrin homology domain and the unique carboxyl-terminal domain. Syntrophin was also able to inhibit actin-activated myosin ATPase activity and actomyosin super-precipitation. alpha-Syntrophin co-localized with cortical F-actin fibers when expressed in Chinese hamster ovary cells, and deletion of the actin-binding region abolished co-localization. Most of exogenous or endogenous syntrophin also co-localized with stress fibers in endothelial and smooth muscle (A7r5) cells. However, syntrophins were mostly localized in the cytosol of serum-starved C2C12 or primary cultured skeletal muscle myotubes, and translocated to the membrane upon treatment with lysophosphatidic acid or the actin-stabilizing agent jasplakinolide. The actin-depolymerizing agent latrunculin-B abolished this syntrophin translocation. These findings suggest that syntrophin is an actin-binding protein the subcellular localization of which is regulated through cytoskeletal reorganization.

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 Ca 2+ was shifted to the left in a parallel manner, suggesting an increase in Ca 2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca 2+ 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. Ca 2+-, K +-EDTA- or Mg 2+-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 Ca 2+ 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.

Turbidity of Myofibril and Actomyosin Suspensions and Its Change Induced by ATP

Optical properties of suspensions of contractile models, myofibrils and two actomyosins (“natural” and “synthetic”), have been investigated. The turbidity and the wavelength exponent of suspensions of the original, contracted, and aggregated models have been measured at different concentrations, light wavelengths, and solid angles of the spectrophotometer photocell. Change in the turbidity of these suspensions upon addition of MgATP is compared to changes in the particle size and gel phase volume of suspensions. The turbidity of myofibril and actomyosin suspensions is shown to be strongly dependent on the conditions of its measurement. The extent of turbidity changes upon the contraction of myofibrils and superprecipitation of actomyosin also depends on measurement conditions and is proportional to the difference between the densities of the original and contracted particles. It is suggested that the increase in suspension turbidity upon addition of MgATP is due to changes in the form of the light-scattering angular diagram rather than to the enhancement of total light scattering of suspensions.

Study on the superprecipitation of cardiac actomyosin of the rat fed on grains from Keshan disease areas

Study on the superprecipitation of cardiac actomyosin of the rat fed on grains from Keshan disease areas

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.

Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recognizes the calmodulin-binding region

The regulatory domain of smooth muscle myosin light chain kinase (MLCK) was studied using monoclonal antibodies. Of the 22 monoclonal antibodies tested, a monoclonal antibody designated LKH-18 was found to activate MLCK in the absence of Ca2+/calmodulin. This activation was even greater when an Fab fragment of LKH-18 was used. Consequently, the actin-dependent smooth muscle myosin ATPase activity and the superprecipitation of actomyosin were significantly activated by MLCK plus LKH-18, even in the absence of Ca2+/calmodulin. The antibody-binding site was studied using proteolytic fragments and synthetic peptide analogues of MLCK. Immunoblot analysis revealed that LKH-18 reacted with the 66 kDa calmodulin-dependent active fragment but not with the 64 kDa inactive fragment or with the 61 kDa calmodulin-independent active fragment. Furthermore, LKH-18 reacted with MLCK-(796-815)-peptide but not with MLCK-(786-801)-peptide or with MLCK-(796-807)-peptide. Therefore the LKH-18-binding site was assigned to amino acid residues 808-815 of MLCK, which are thought to be a part of the calmodulin-binding site. The present results suggest that the binding of ligand to this region induces a conformation change in MLCK and that this abolishes the action of the inhibitory region which exists next to the N-terminal side of the calmodulin-binding site.

Molecular Pharmacology of Calcium, Calmodulin-Dependent Myosin Phosphorylation in Vascular Smooth Muscle

The Ca2+, calmodulin (CaM)-dependent phosphorylation of the 20 kDa myosin light chain (LC20) is accepted as an important component of the regulatory mechanism in smooth muscle contraction. Since we have originally developed selective inhibitors of each process of the intracellular Ca2+ messenger system, the effect of a newly synthesized compound ML-9, a myosin light chain kinase (MLCK) inhibitor on superprecipitation of actomyosin, isometric tension development and phosphorylation of LC20 in vascular smooth muscle was investigated. Superprecipitation of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscle cells of the rabbit mesenteric artery, ML-9 inhibited both Ca2+ and Ca2+, CaM-independent MLCK-induced contraction. In the intact vascular strips, increase in LC20 phosphorylation reached a maximal value within 10 sec from a resting value, and then declined to near the basal level during the maintained isometric force developed in response to 50 mmol/L KCl. Both the maximal rate and extent of KCl-induced contraction and the phosphorylation of LC20 were also inhibited by ML-9. It antagonized the contraction induced by various contractile agonists, such as NE, 5HT, His, and Ang II concomitant with the inhibition of LC20 phosphorylation. These results suggest that ML-9 inhibits the actin-myosin interaction through the modulation of LC20 phosphorylation via the inhibition of MLCK activity. ML-9 will aid in determining pathophysiological functions of MLCK of increased vascular contractility in hypertension.

Inhibition of cardiovascular low‐Km cAMP phosphodiesterase activity by medorinone

AbstractMedorinone (5‐methyl‐1, 6‐naphthyridin‐2(1H)‐one) is a structurally novel cardiotonic/vasodilator agent. The purpose of the present study was to evaluate the effects of medorinone on subcellular systems involved in the contractile‐relaxation processes of cardiac and vascular smooth muscle (VSM); cyclic nucleotide phosphodiesterase (PDE), Ca2+‐regulated contractile proteins, and sarcolemmal Na+‐K+ ATPase. Medorinone produces concentration‐related inhibition (IC50 values between 0.38 and 1.3 μ) of the cGMP‐inhibitable, low‐Km cAMP PDE (peak III PDE from a DEAE‐cellulose column) present in guinea pig and canine cardiac and vascular smooth muscle. The potency of medorinone is similar to that of milrinone and papaverine, less than CI‐930, and greater than imazodan, piroximone, and amrinone. Medorinone is approximately 300–400 × less potent an inhibitor of the low‐Km cAMP PDE. Medorinone appears to be a non‐linear mixed inhibitor of the low‐Km cAMP PDE having a Ki value of approximately 0.3 μM. Medorinone is also an equipotent inhibitor (IC50 values between 0.23 and 0.35 μM) of the low‐m cAMP PDE present in soluble and particulate fractions obtained from normal and myopathic human hearts, with no differences in potency observed between normal and myopathics. Medorinone (10 and 100 μM) does not affect VSM myosin light chain phosphorylation or VSM actomyosin superprecipitation. Moreover, medorinone does not affect cardiac myofibrillar ATPase activity or cardiac sarcolemmal Na+‐K+ ATPase activity. In summary, these data are consistent with the hypothesis that inhibition of the low‐Km cAMP PDE isozyme by medorinone in cardiac and vascular smooth muscle is a mechanism by which this agent produce both positive cardiac inotropy and VSM relaxation.

A novel peptide inhibitor of the myosin ATPase from an Okinawan marine sponge

A substance which inhibits the myosin ATPase has been detected from an Okinawan marine sponge. The inhibitor has been isolated from the methanol-soluble extract of the sponge by gel filtration and hydrophobic chromatography. The isolated inhibitor is an amphipathic peptide, which is rich in Asp, Glu, Ser, and Gly, and devoid of Met and Trp. The molecular weight of the peptide is about 6300 as determined by gel filtration, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis. The peptide is a potent inhibitor not only for the K+-, Ca2+-, and Mg2+-ATPases of myosin, its subfragment-1, and actomyosin, but also for superprecipitation of actomyosin, inhibiting them completely in the range of 10-400 ng/ml. The peptide inhibitor may provide a useful tool to elucidate the structure-function relationship of the myosin ATPase.

Inhibition of calmodulin and protein kinase C by amiodarone and other class III antiarrhythmic agents

Class III antiarrhythmic agents may prolong refractoriness via modulation of ion channels, which may be sensitive to Ca2+ regulatory proteins or enzymes. Accordingly, the purpose of this study was to quantitate the effects of several structurally diverse class III antiarrhythmic agents on calmodulin-regulated enzymes and protein kinase C activity, and to evaluate the ability of these agents and known calmodulin antagonists to prolong cardiac refractoriness in vivo. The rank order of potency (IC50;microM) of selected class III antiarrhythmic agents and reference calmodulin antagonists as inhibitors of calmodulin-regulated phosphodiesterase activity were: calmidazolium (0.12 microM) greater than amiodarone (0.62 microM) greater than desethylamiodarone (1.5 microM) greater than trifluoperazine (4.3 microM), bepridil (5 microM) greater than W-7 (7.5 microM), clofilium (13 microM). Similar concentration-related inhibition was evident in a second calmodulin-regulated system, inhibition of myosin light-chain phosphorylation and superprecipitation of arterial actomyosin. Sotalol and tetraethylammonium were inactive at 100 microM. Protein kinase C activity was also inhibited by some of these agents; desethylamiodarone (IC50 = 11 microM) was more potent than the reference agent, H-7 (IC50 = 79 microM), or amiodarone (38% inhibition at 100 microM) and clofilium (32% inhibition at 100 microM). In vivo, the minimally effective doses required to increase ventricular effective refractory periods in paced guinea pigs were (in mg/kg) bepridil, sotalol [1] greater than clofilium [3] greater than amiodarone [10] greater than W-7, desethylamiodarone [20]. No changes in refractory period were noted with maximum testable doses of calmidazolium or trifluoperazine. These studies show that some, but not all, class III antiarrhythmic agents are effective and potent calmodulin antagonists or protein kinase C inhibitors. Moreover, some calmodulin antagonists are effective at prolonging refractoriness in vivo. However, a lack of correlation between these agents suggests that these mechanisms are not solely responsible for the prolongation of refractoriness of all class III agents.

Anti-Gizzard MLCK Monoclonal Antibody MM13 Inhibits Superprecipitation and Phosphorylation of Bovine Aortic Smooth Muscle Actomyosin1

We prepared monoclonal antibodies directed against chicken gizzard myosin light chain kinase (MLCK) and used them to study the contractile system of aortic smooth muscle. One monoclonal antibody, MM13, dose dependently inhibited actomyosin superprecipitation of bovine aortic smooth muscle, in accord with the suppression of 20 kDa myosin light chain phosphorylation by endogenous kinase. Immunoblotting analysis demonstrated that MM13 cross-reacted with the 150,000 Mr peptide of bovine aortic actomyosin preparation. The bovine aortic MLCK was purified approximately 2,400-fold to apparent homogeneity by three steps of column chromatography. The purified enzyme has a molecular weight of 150,000 and a slower mobility than chicken gizzard MLCK (130,000 Mr), as determined by SDS-polyacrylamide gel electrophoresis. MM13 also cross-reacted with purified bovine aortic MLCK and inhibited the kinase activity, in vitro. We interpret these findings to mean that binding of the anti-gizzard MLCK monoclonal antibody directly to aortic smooth muscle MLCK (150,000 Mr) decreases the phosphorylation of the 20 kDa myosin light chain, thus suppressing the aortic smooth muscle myosin-actin interaction.

Antihypertensive compounds with potent protein kinases inhibitory activity

A new indorocarbazole antibiotic SF2370 has been found in the culture broth of Actinomadura sp. ; it has also been found to have the activities of antihypertension and diuresis. Derivatives of SF2370, NA0344, NA0345 and NA0346, when injected (0.1−1 mg/kg i.v.) significantly lowered the blood pressure of anesthetized normotensive rats and showed a long lasting antihypertensive action. In the case of p.o. administration to conscious restrained SHRs, measurement of blood pressure by the plethysmographic method indicated that 10 mg/kg of the compounds was enough to lower the blood pressure; the antihypertensive activities were found to remain more than 12 hrs after oral administration. By studying of the mode of antihypertensive action, these compounds dose dependently relaxed the isolated aortic preparation contracted by norepinephrine. In addition, it was found that protein kinase C activity of mice brain was inhibited by the compounds; the IC 50 values were in the range of 0.062 – 0.20 μM. Moreover, a superprecipitation of actomyosin from smooth muscle of chicken gizzard was inhibited by the compounds, having an IC 50 values of 0.31 – 0.72 μM.

Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA‐ATPase that enhanced the superprecipitation of actomyosin

1. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 microM purealin. 2. The ATPase activity of myosin B was also elevated to 160% of the control value by 10 microM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+-ATPase), and the concentration for the half inhibition was 4 microM. 3. On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA-ATPase). The maximum activation by 10 microM purealin was 160% of the control value. 4. Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment-1 instead of myosin. 5. These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA-ATPase and actomyosin ATPase of myosin B.

Calpain abolishes the effect of filamin on the actomyosin system in platelets

Platelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of filamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca 2+, filamin loses its ability to cross-link F-actin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for the contraction of the activated platelets.

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 Ca 2+- and Mg 2+-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.

Ca2+-independent gizzard myosin light chain kinase produced by cross-linking of the enzyme with calmodulin using glutaraldehyde.

Cross-linked complex of gizzard myosin light chain kinase (MLCK) and calmodulin (CM) was produced by glutaraldehyde treatment of a mixture of these proteins in a high Ca2+ (0.1 mM) solution. Although the specific activity was reduced, this complex showed MLCK activity in a Ca2+-independent manner, different from the original MLCK whose activity was Ca2+-dependent. Chlorpromazine, one of the CM antagonists, was no longer able to inhibit the MLCK activity of this complex. These observations support the previously proposed hypothesis on the regulatory mechanism of MLCK activity via Ca2+. This complex could be regarded as another kind of Ca2+-independent MLCK different from that obtained by chymotryptic digestion of MLCK (Walsh, M.P., Dabrowska, R., Hinkins, S., & Hartshorne, D.J. (1982) Biochemistry 21, 1919-1925). This complex caused superprecipitation of gizzard actomyosin and enhanced actin-activated ATPase of myosin Ca2+-independently.