scholarly journals Modification of the interactions of myosin with actin and 5′-adenylyl imidodiphosphate by substitution of ethylene glycol for water

1984 ◽  
Vol 217 (1) ◽  
pp. 169-177 ◽  
Author(s):  
S B Marston ◽  
R T Tregear
Keyword(s):  
Ethylene Glycol ◽  
Dissociation Rate ◽  
Detectable Effect ◽  
Muscle Fibres ◽  
Skeletal Muscle Myosin ◽  
Specific Effects ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Glycerinated Muscle ◽  
Myosin X

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5′-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.

Contractile proteins from the tomato

1980 ◽  
Vol 58 (7) ◽  
pp. 797-801 ◽  
Author(s):  
Maryanne Vahey ◽  
Stylianos P. Scordilis
Keyword(s):  
Ionic Strength ◽  
Ethylenediaminetetraacetic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Sds Page ◽  
Myosin Subfragment 1 ◽  
Parenchymal Cells ◽  
Low Ionic Strength

Proteins exhibiting all of the basic structural and biochemical characteristics of actin and myosin have been isolated from the parenchymal cells of the fruit of the tomato, Lycopersicon esculentum. Crude cytoplasmic extracts of these cells contain filaments that can be decorated by rabbit skeletal muscle myosin subfragment-1 (S-1). Polymerized tomato actin activates the Mg2+–ATPase of both skeletal and tomato myosin at physiological ionic strength. Tomato actin comigrates with skeletal actin on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) indicating an apparent molecular weight of 45 000. High ionic strength extracts of tomato contain a myosin whose ATPase activity in 0.5 M KCl is maximal in the presence of K+-ethylenediaminetetraacetic acid (K+-EDTA) and is inhibited by Mg2+. Tomato myosin interacts with skeletal F-actin to form an actomyosin complex that can be dissociated by ATP. At low ionic strength the Mg2+–ATPase of the myosin can be activated by actin.

scholarly journals Correlation between calponin and myosin subfragment 1 binding to F-actin and ATPase inhibition

1997 ◽  
Vol 321 (2) ◽  
pp. 519-523 ◽  
Author(s):  
Pawel T. SZYMANSKI ◽  
Zenon GRABAREK ◽  
Terence TAO
Keyword(s):  
Troponin I ◽  
Sequence Similarity ◽  
Actin Binding ◽  
Amino Acid Sequence Similarity ◽  
Smooth Muscle Contractility ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin ◽  
Atpase Inhibition

Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the present work we have examined the effect of recombinant chicken gizzard α-calponin (RαCaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one RαCaP molecule to every three to four actin monomers is sufficient for maximal inhibition of actoŐS1 ATPase. At this RαCaP/actin ratio RαCaP does not interfere with S1 binding to F-actin. At higher concentrations, RαCaP displaces S1 from F-actin and a 1:1 RαCaPŐactin monomer complex is formed. RαCaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between RαCaP and troponin I in their actin-binding regions.

scholarly journals Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation

2020 ◽  
Vol 21 (12) ◽  
pp. 4421
Author(s):  
Yurii S. Borovikov ◽  
Armen O. Simonyan ◽  
Stanislava V. Avrova ◽  
Vladimir V. Sirenko ◽  
Charles S. Redwood ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Muscle Weakness ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Spatial Arrangement ◽  
Muscle Fibres ◽  
Thin Filaments ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

The actomyosin ATPase: a two-state system

1992 ◽  
Vol 336 (1276) ◽  
pp. 63-71 ◽  
Keyword(s):  
Equilibrium Constants ◽  
Muscle Fibres ◽  
Close Coupling ◽  
Contracting Muscle ◽  
Myosin Subfragment ◽  
Association Reaction ◽  
Myosin Subfragment 1 ◽  
Series Of Experiments ◽  
Contraction Cycle ◽  
Key Events

Studies of the interaction between actin and myosin subfragment 1 (S1) in solution have shown that the association reaction takes place in at least two steps. Initially the association is relatively weak to form a complex called the A state which can then isomerize to the R state. The rate and equilibrium constants for the isomerization have been measured and are shown to depend upon the nucleotide bound to the SI ATPase site; with ATP bound the A state is preferred but as ATP is hydrolysed and the products are sequentially released then the complex gradually shifts to the A state. An extensive series of experiments have characterized the A-to-R isomerization both in solution and in contracting muscle fibres and have shown it to be closely associated with the key events in the ATP-driven contraction cycle: the conformational change from the A to the R state can be monitored by fluorescent probes on either actin or the nucleotide; the isomerization can be perturbed by increases in hydrostatic pressure; the actin-induced acceleration of the rate of product release from myosin is coupled to the A-to-R isomerization; tropomyosin may control actin and myosin interaction by controlling the ismoerization step and finally pressure perturbations of contracting muscle fibres shows there to be a close coupling between the isomerization of acto.S1 and the force generating event of muscle contraction.

Skeletal muscle myosin subfragment 1 dimers

1997 ◽  
Vol 65 (1) ◽  
pp. 85-90 ◽  
Author(s):  
Karen Claire ◽  
Robert Pecora ◽  
Stefan Highsmith
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin

scholarly journals Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method

1983 ◽  
Vol 209 (3) ◽  
pp. 617-626 ◽  
Author(s):  
T E Barman ◽  
D Hillaire ◽  
F Travers
Keyword(s):  
Ethylene Glycol ◽  
Myosin Atpase ◽  
Experimental Conditions ◽  
Binding Process ◽  
Atp Concentration ◽  
Myosin Subfragment ◽  
Rapid Flow ◽  
Myosin Subfragment 1 ◽  
Ph Decrease ◽  
Quench Method

1. The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing [gamma-32P]ATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.

scholarly journals The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin

1985 ◽  
Vol 232 (2) ◽  
pp. 343-349 ◽  
Author(s):  
A H Criddle ◽  
M A Geeves ◽  
T Jeffries
Keyword(s):  
Dissociation Rate ◽  
Actin Binding ◽  
Heavy Meromyosin ◽  
Pyrene Fluorescence ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Two Phases

A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.

scholarly journals Characterization of an actin-myosin head interface in the 40–113 region of actin using specific antibodies as probes

1990 ◽  
Vol 271 (2) ◽  
pp. 407-413 ◽  
Author(s):  
J P Labbé ◽  
C Méjean ◽  
Y Benyamin ◽  
C Roustan
Keyword(s):  
Myosin Head ◽  
Interface Model ◽  
Filamentous Actin ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Myosin Interaction ◽  
Muscle Myosin ◽  
Covalent Complex

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the ‘multisite interface model’, where the two actin sites could modulate each other during S-1 interaction.

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