Changes in the State of Actin during Superprecipitation of Actomyosin

Strzelecka‐Gołaszewska, Hanna; Drabikowski, W.; Jakubiak, Miroslawa

doi:10.1111/j.1432-1033.1975.tb02154.x

Cited by 55 publications

(34 citation statements)

References 40 publications

(26 reference statements)

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“…As shown in Table 1, all preparations, either fresh or stored in the G form at 0 'C in the presence of 0.2 mM ATP, 0.2 mM CaC12 for at least 48 h, contained about 1 mole of bound adenine nucleotide per mole of actin monomer. The content of tightly bound divalent cation determined by EDTA titration was also 1 mole/mole as has been found for skeletal muscle actin [13,21]; since the amounts of bound Mg2+, determined by atomic absorption, were found to be negligible in all three kinds of gizzard preparations, it may be supposed that the bound cation is Ca2+. The results presented in Table 1 also show that the presence of free Ca2+ is necessary to protect gizzard G-actin against denaturation.…”

Section: Resultsmentioning

confidence: 91%

“…1, gels A l , B1, Cl). In the case of actin from skeletal muscle, the removal of the main contaminant, tropomyosin, may be achieved by the extraction of the acetone-dried muscle powder at 0°C [7] and the use of either low (30 mM) or relatively high (0.6 M) concentrations of KCl for the polymerization of actin during its purification [17,18]. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The total concentration of the bound divalent cation was determined by spectrophotometric microtitration with EDTA as described previously [13] and that of Mg2+ was measured in an EEL 240 atomic absorption spectrophotometerin the presence of 1 % L a c 4 to eliminate the interference by phosphates.…”

Section: Analytical Proceduresmentioning

confidence: 99%

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Chicken‐Gizzard Actin: Polymerization and Stability

Strzelecka‐Gołaszewska

Próchniewicz

Nowak

et al. 1980

European Journal of Biochemistry

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Preparations of chicken gizzard actin obtained from acetone-dried muscle powders prepared with various methods developed for skeletal muscle contain variable amounts of a P-actinin-like protein. This contamination is minimized if the procedure of muscle powder preparation includes washing with EDTA solution, and can be completely removed by gel filtration of G-actin on Sephadex G-100. The presence of 8-actinin activity manifests itself in an increased rate of actin polymerization, low filament lengths resulting in low reduced viscosity and enhanced ATP-splitting activity of actin polymer, and instability of the polymer in the absence of free ATP. Gizzard actin purified on a Sephadex G-100 column does not differ from rabbit skeletal muscle actin in its polymerization properties. The distinct property of gizzard actin is the instability of its G form in the absence of added Ca2+, indicating that the affinity of this cation for the single high-affinity site in gizzard actin is lower than in skeletal muscle actin.The procedures generally used to isolate muscle actin involve preparation of acetone-dried muscle powder from which monomeric actin is extracted and purified by reversible polymerization combined with ultracentrifugation. Earlier observations [l -31 indicated that polymerizable actin can be obtained from various vertebrate smooth muscles if the acetone-dried powder is prepared with the method of Carsten and Mommaerts [4]

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

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Chicken‐Gizzard Actin: Polymerization and Stability

Strzelecka‐Gołaszewska

Próchniewicz

Nowak

et al. 1980

European Journal of Biochemistry

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“…Troponin Complex Formation-Rabbit skeletal F-actin was prepared as described by Strzelecka-Golaszewska et al (19). Porcine cardiac myosin was purified according to Murakami et al (20).…”

Section: Methodsmentioning

confidence: 99%

Different Functional Properties of Troponin T Mutants That Cause Dilated Cardiomyopathy

Venkatraman

Harada

Gomes

et al. 2003

Journal of Biological Chemistry

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The effects of Troponin T (TnT) mutants R141W and ⌬K210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca 2؉ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-⌬K210 caused a significant decrease in Ca 2؉ sensitivity, whereas the TnT-R141W did not result in any change in Ca 2؉ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-⌬K210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-⌬K210 mutant decreased maximal ATPase activity in the presence of Ca 2؉ . However, the TnT-K273E mutation caused a significant increase in Ca 2؉ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca 2؉ sensitivity is not the only determinant for the phenotype of DCM.

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“…Actin was prepared from acetone-dried muscle powder of rabbit skeletal muscles as described previously [66]. Centrifuged F-actin pellets were resuspended in a solution containing 1 mM MgCI2 and 50 mM Hepes buffer, pH 8.0, directly before use.…”

Section: Protein Preparationsmentioning

confidence: 99%

Conformational transitions in the myosin head induced by temperature, nucleotide and actin

Rędowicz

Szilágyi

Strzelecka‐Gołaszewska

1987

European Journal of Biochemistry

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Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (Sl) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides.The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its P,yimido analog AdoPP[NH]P) at 25 "C were observed when the digestion was carried out at 0°C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoPP[NH]P at 0°C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.It is generally accepted that contraction of muscle results from a relative sliding movement of the myosin thick filaments past the actin thin filaments driven by a cyclic interaction of myosin heads or cross-bridges with actin, each cycle being coupled to hydrolysis of one molecule of ATP [l]. However, the detailed mechanism of conversion of the free energy of ATP hydrolysis into mechanical energy remains a subject of debate. A prevailing model suggested by H. E. Huxley [l] and by A. F. Huxley and Simmons [2] assumes that force is generated by an active change in the angle of attachment of myosin heads to actin filaments by rotation about a flexible joint between the head (myosin subfragment-1) and the neck region (subfragment-2) of the cross-bridge. An alternative proposal is the rotation of one portion or domain of the head relative to the other portion which remains rigidly attached to actin [3,4]. The latter possibility is in better agreement with the evidence from recent X-ray diffraction studies on muscle [5 -71 and with fluorescence polarization [8] and EPR studies [9] on glycerinated muscle fibers.The possible existence of domains within the myosin head has been inferred from limited proteolysis studies on myosin subfragment-1. The heavy chain of chymotryptic S1 of myosin from rabbit skeletal muscle is split by trypsin into three major fragments with molecular masses of 27 kDa, 50 kDa, and 20 kDa [lo, 111 which are aligned in this order relative to the NH2-terminus of the heavy chain [12]. Two heavy-chain segments (about ...

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Changes in the State of Actin during Superprecipitation of Actomyosin

Cited by 55 publications

References 40 publications

Chicken‐Gizzard Actin: Polymerization and Stability

Chicken‐Gizzard Actin: Polymerization and Stability

Different Functional Properties of Troponin T Mutants That Cause Dilated Cardiomyopathy

Conformational transitions in the myosin head induced by temperature, nucleotide and actin

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