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 The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

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.

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 Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin

1999 ◽  
Vol 263 (2) ◽  
pp. 396-401 ◽  
Author(s):  
Sumiko Kimura ◽  
Akira Ichikawa ◽  
Junko Ishizuka ◽  
Shiho Ohkouchi ◽  
Takei Kake ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Rabbit Skeletal Muscle ◽  
Filament Formation

scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2018 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Regulatory Proteins ◽  
Actin Assembly ◽  
Cryo Electron Microscopy ◽  
Pointed End ◽  
Binding Loop

AbstractWe used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5’-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 Å, 3.4 Å and 3.6 Å. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.Significance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the γ-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum

1998 ◽  
Vol 274 (6) ◽  
pp. C1738-C1744 ◽  
Author(s):  
Leopoldo De Meis
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Heat Production ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Heparin Sodium ◽  
The Difference

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201–204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (Δ H cal) value found with both rabbit and trout varied between −10 and −12 kcal/mol in leaky vesicles (no gradient) and between −20 and −22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of Δ H cal measured with and without gradient was detected in the range of 30–35°C and disappeared when the temperature was decreased below 30°C. For the trout, the difference was detected between 20 and 25°C and disappeared below 20°C. The effect of the gradient on the Δ H cal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.

Actin and myosin: control of filament assembly

1982 ◽  
Vol 299 (1095) ◽  
pp. 247-261 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thick Filament ◽  
Light Chains ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
Filament Formation ◽  
Tail Region ◽  
Filament Assembly ◽  
Fluorescence Energy

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35 S-labelled actin monomers into unlabelled actin filaments, and exchange of [ 14 C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca 2+ -dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.

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