scholarly journals Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

2020 ◽  
Vol 21 (22) ◽  
pp. 8720
Author(s):  
Marina A. Marchenko ◽  
Victoria V. Nefedova ◽  
Daria S. Yampolskaya ◽  
Galina V. Kopylova ◽  
Daniil V. Shchepkin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Fine Tuning ◽  
Actin Binding ◽  
Sliding Velocity ◽  
Scanning Calorimetry ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
High Flexibility ◽  
Thermal Stability Of

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca2+ concentration. However, the mutations differently affected the Ca2+ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca2+-dependent interaction of myosin heads with F-actin.

scholarly journals Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles

2021 ◽  
Vol 22 (7) ◽  
pp. 3466
Author(s):  
Svetlana V. Klinova ◽  
Boris A. Katsnelson ◽  
Ilzira A. Minigalieva ◽  
Oksana P. Gerzen ◽  
Alexander A. Balakin ◽  
...  
Keyword(s):  
Cadmium Oxide ◽  
Male Rats ◽  
Sliding Velocity ◽  
Muscle Type ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Toxic Impact ◽  
Cdo Nanoparticles

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO–NP and PbO–NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling β–MHC. The type of CdO–NP + PbO–NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb–NP and CdO–NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution

1992 ◽  
Vol 263 (2) ◽  
pp. H464-H472 ◽  
Author(s):  
H. Yamashita ◽  
S. Sugiura ◽  
T. Serizawa ◽  
T. Sugimoto ◽  
M. Iizuka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Motility Assay ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Actin Cables

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

scholarly journals Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

2020 ◽  
Author(s):  
Venukumar Vemula ◽  
Tamas Huber ◽  
Marko Usaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Cellular Motility ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
In Vitro Motility ◽  
Filament Dynamics

AbstractActin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] ∼6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

scholarly journals CHANGES OF MYOCARDIUM CONTRACTILITY ASSOCIATED WITH A SUBCHRONIC LEAD INTOXICATION IN RATS

2020 ◽  
Vol 99 (2) ◽  
pp. 193-199
Author(s):  
Svetlana V. Klinova ◽  
Y. L. Protsenko ◽  
O. N. Lookin ◽  
A. A. Balakin ◽  
L. V. Nikitina ◽  
...  
Keyword(s):  
Contractile Function ◽  
Intraperitoneal Administration ◽  
Male Rats ◽  
Lead Intoxication ◽  
Mechanical Activity ◽  
Papillary Muscles ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

Introduction. There is a high chance of a link between cardiovascular conditions and occupational or environmental exposure to lead. Taking into account the peculiarities of lead intoxication and the metal common occurrence it appeared to necessarily prove further experimental research of lead cardiotoxicity. Material and methods. After repeated intraperitoneal administration of sublethal doses of lead acetate to outbred male rats 3 times a week for 5 weeks, there was obtained the moderately pronounced subchronic lead intoxication manifested by some characteristic features. Cardiotoxic effects on myocardial contractility were studied by the analysis of the mechanical activity of isolated preparations of right ventricular trabeculae and papillary muscles contracting in isotonic and physiological modes of loading. Myocardial contractile function was also studied at the molecular level by measuring the sliding velocity of reconstructed thin filaments over myosin. Results. In papillary muscles lead intoxication led to a decrease in the maximal rate of isotonic shortening for all afterloads and a decrease in the thin filament sliding velocity in the in vitro motility assay. The same type of muscle from lead-exposed rats displayed marked changes in most of the main characteristics of afterload contraction-relaxation cycles, but in trabeculae, these changes were less pronounced. The reported changes were attenuated to some extent in rats similarly exposed to lead while being treated with a Ca-containing bio protector. The amount of work produced by both muscle preparations was unchanged under lead intoxication over the entire range of afterloads, which is an evidence of adaptation to the production of adequate mechanical work despite resulting contractility disturbances. Conclusions. 1. Subchronic lead intoxication was shown to cause contractile dysfunction of rat myocardium. In papillary muscles the alterations were observed more than in trabeculae. The changes in contractile proteins corresponded with those seen in myocardium structures. 2. The reported changes were attenuated to some extent in rats being treated with a Ca-containing bio protector.

Phosphorylation of calponin in airway smooth muscle

1997 ◽  
Vol 272 (1) ◽  
pp. L115-L123 ◽  
Author(s):  
J. Pohl ◽  
S. J. Winder ◽  
B. G. Allen ◽  
M. P. Walsh ◽  
J. R. Sellers ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Inhibitory Effect ◽  
Tracheal Smooth Muscle ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Kinase C ◽  
Gradient Gel Electrophoresis

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.

scholarly journals Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca 2+-sensitivity of Actin-Myosin Interaction

Acta Naturae ◽  
2013 ◽  
Vol 5 (3) ◽  
pp. 126-129 ◽  
Author(s):  
D. V. Shchepkin ◽  
A. M. Matyushenko ◽  
G. V. Kopylova ◽  
N. V. Artemova ◽  
S. Y. Bershitsky ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Myosin Head ◽  
Motility Assay ◽  
Published Data ◽  
Physiological Effects ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Myosin Interaction

We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са 2+ concentrations and the Са 2+-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tmmyosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.

A model for the coregulation of smooth muscle actomyosin by caldesmon, calponin, tropomyosin, and the myosin regulatory light chain

1994 ◽  
Vol 72 (11) ◽  
pp. 1400-1409 ◽  
Author(s):  
Joe R. Haeberle ◽  
Mark E. Hemric
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Regulatory Protein ◽  
Regulatory Light Chain ◽  
Motility Assay ◽  
Actin Binding ◽  
Shortening Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

The purpose of these studies was to evaluate the effects of the actin-binding proteins tropomyosin, caldesmon, and calponin on the activation of smooth muscle actomyosin by phosphorylation of the regulatory light chain of myosin (LC20), and to interpret these findings in the context of a two-state kinetic model for the cross-bridge cycle. An in vitro motility assay was used to broadly classify each regulatory protein according to whether it modulates the apparent on-rate for cross bridges (fapp) or the apparent off-rate (gapp). In addition to measuring actin-filament velocity, a method was developed to measure relative changes in the force exerted on actin filaments under isometric conditions. Based primarily on the results of these motility studies, a qualitative model is proposed in which LC20 phosphorylation, tropomyosin, and caldesmon all regulate fapp and calponin regulates gapp. The model predicts that the sensitivity of activation by LC20 phosphorylation is determined by tropomyosin, caldesmon, and calponin, whereas unloaded shortening velocity is regulated primarily by calponin.Key words: smooth muscle, caldesmon, calponin, tropomyosin, motility assay.

scholarly journals Mechanism of the Allosteric Interaction of Tropomyosin (TM) and Actin to Increase the Sliding Velocity and Force as Measured by In-Vitro Motility Assay using α-Tm Mutants

2011 ◽  
Vol 100 (3) ◽  
pp. 359a-360a
Author(s):  
Yusuke Oguchi ◽  
Junji Ishizuka ◽  
Sarah E. Hitchcock-DeGregori ◽  
Shin’ichi Ishiwata ◽  
Masataka Kawai
Keyword(s):  
Motility Assay ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Allosteric Interaction

Thermal Stability of N-Heterocycle-Stabilized Iodanes – A Systematic Investigation

2019 ◽  
Author(s):  
Andreas Boelke ◽  
Yulia A. Vlasenko ◽  
Mekhman S. Yusubov ◽  
Boris Nachtsheim ◽  
Pavel Postnikov
Keyword(s):  
Thermal Stability ◽  
Differential Scanning Calorimetry ◽  
Thermogravimetric Analysis ◽  
Systematic Investigation ◽  
Scanning Calorimetry ◽  
Thermal Stability Of

<p>The thermal stability of pseudocyclic and cyclic <i>N</i>-heterocycle-stabilized (hydroxy)aryl- and mesityl(aryl)-l<sup>3</sup>-iodanes (NHIs) through thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) is investigated. NHIs bearing <i>N</i>-heterocycles with a high N/C-ratio such as triazoles show among the lowest descomposition temperatures and the highest decomposition energies. A comparison of NHIs with known (pseudo)cyclic benziodoxolones is made and we further correlated their thermal stability with reactivity in a model oxygenation. </p>

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