scholarly journals Isolation of Nebulin from Rabbit Skeletal Muscle and Its Interaction with Actin

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Ryo Chitose ◽  
Atsushi Watanabe ◽  
Masato Asano ◽  
Akira Hanashima ◽  
Kouhei Sasano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Column Chromatography ◽  
Salt Solution ◽  
Actin Filaments ◽  
Thin Filament ◽  
Full Length ◽  
Rabbit Skeletal Muscle ◽  
Native Form ◽  
20 Nm ◽  
Vertebrate Skeletal Muscle

Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin,α-actinin,β-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.

scholarly journals Ca2+ Regulation of Rabbit Skeletal Muscle Thin Filament Sliding: Role of Cross-Bridge Number

2003 ◽  
Vol 85 (3) ◽  
pp. 1775-1786 ◽  
Author(s):  
Bo Liang ◽  
Ying Chen ◽  
Chien-Kao Wang ◽  
Zhaoxiong Luo ◽  
Michael Regnier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Rabbit Skeletal Muscle ◽  
Cross Bridge ◽  
Muscle Thin Filament ◽  
Bridge Number

scholarly journals Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

2007 ◽  
Vol 579 (2) ◽  
pp. 313-326 ◽  
Author(s):  
Alicia Moreno-Gonzalez ◽  
Todd E. Gillis ◽  
Anthony J. Rivera ◽  
P. Bryant Chase ◽  
Donald A. Martyn ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Rabbit Skeletal Muscle ◽  
Muscle Fibres ◽  
Thin Filament Regulation ◽  
Skeletal Muscle Fibres

The structure of actin filaments and the origin of the axial periodicity in the I -substance of vertebrate striated muscle

1964 ◽  
Vol 160 (981) ◽  
pp. 449-460 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscope ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Rabbit Skeletal Muscle ◽  
Contractile Apparatus ◽  
Detailed Structure

Certain advances due mainly to H. E. Huxley (see Huxley 1961, 1963) have made it possible to use the electron microscope to study the detailed structure of the filaments in the contractile apparatus. The results of our work on actin filaments have already been published (Hanson & Lowy 1962, 1963). We shall now examine some of the consequences of these findings, including certain unsolved problems which they raise. Actin in the polymerized form ( F -actin) has been prepared from rabbit skeletal muscle by the usual methods and examined in negatively stained preparations in the electron microscope (Hanson & Lowy 1963). It has been found that solutions of F -actin are, in fact, suspensions of filaments. These consist of globular subunits arranged in a characteristic helical manner (figure 15).

scholarly journals Mechanism of K+-induced actin assembly.

1982 ◽  
Vol 93 (3) ◽  
pp. 648-654 ◽  
Author(s):  
J D Pardee ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Dictyostelium Discoideum ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Uv Spectroscopy ◽  
Rabbit Skeletal Muscle ◽  
Actin Assembly ◽  
Filament Formation ◽  
Monomeric Species ◽  
Rapid Formation

The assembly of highly purified actin from Dictyostelium discoideum amoebae and rabbit skeletal muscle by physiological concentrations of KCI proceeds through successive stages of (a) rapid formation of a distinct monomeric species referred to as KCI-monomer, (b) incorporation of KCI-monomers into an ATP-containing filament, and (c) ATP hydrolysis that occurs significantly after the incorporation event. KCI-monomer has a conformation which is distinct from that of either conventional G- or F-actin, as judged by UV spectroscopy at 210-220 nm and by changes in ATP affinity. ATP is not hydrolyzed during conversion of G-actin to KCI-monomer. KCI-monomer formation precedes filament formation and may be necessary for the assembly event. Although incorporation of KCI-monomers into filaments demonstrates lagphase kinetics by viscometry, both continuous absorbance monitoring at 232 nm and rapid sedimentation of filaments demonstrate hyperbolic assembly curves. ATP hydrolysis significantly lags the formation of actin filaments. When half of the actin has assembled, only 0.1 to 0.2 mole of ATP are hydrolyzed per mole of actin present as filaments.

scholarly journals Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

2021 ◽  
Vol 22 (8) ◽  
pp. 4036
Author(s):  
Małgorzata Śliwinska ◽  
Katarzyna Robaszkiewicz ◽  
Piotr Wasąg ◽  
Joanna Moraczewska
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thin Filament ◽  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Troponin Complex ◽  
Increased Sensitivity

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin–myosin interactions.

Electron microscopy of myosin molecules from muscle and non-muscle sources

1976 ◽  
Vol 193 (1110) ◽  
pp. 45-53 ◽  
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Blood Platelets ◽  
Tail Length ◽  
Glycerol Solution ◽  
Mica Substrate ◽  
Absolute Value ◽  
Replication Technique ◽  
20 Nm ◽  
Vertebrate Skeletal Muscle

Myosin molecules from adult and embryonic vertebrate skeletal muscle, vertebrate cardiac muscle, vertebrate smooth muscle, invertebrate muscle, blood platelets and brain have been examined by a modification of Hall’s mica-replication technique in which droplets of myosin in ammonium formate and glycerol solution are sprayed on a mica substrate at low temperature and then dried in vacuum prior to uni-directional shadowing with platinum. Myosin molecules from all these sources are morphologically indistinguishable and have two globular heads joined to a tail whose length does not differ by more than 10 nm from species to species. The absolute value of the tail length is 150 ± 20 nm (a larger error is given because of the difficulty in defining the point where the tail divides to give the two heads).

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