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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 β-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 β-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. [ABSTRACT FROM AUTHOR]

The interaction of tropomyosin with F-actin was studied under various conditions. The maximum ratio of tropomyosin bound to actin was found by ultracentrifugation to be 1:2, by weight. Viscometric study showed, however, no further increase of viscosity due to formation of the actin-tropomyosin complex when the above ratio exceeded 1:5. Higher ionic strength caused dissociation of the complex. The comparison of the rate of polymerization of tropomyosin free actin under various salt conditions with that in the presence of tropomyosin showed that the latter partially modified some properties of actin. The pattern of polymerization of actin extracted at room temperature (RT-actin) resembled that of the mixtures of actin with tropomyosin, although some differences were observed indicating tighter binding in the case of RT-actin, possibly due to the presence of troponin. The results obtained are discusssed from the point of view of the possible localization of tropomyosin in thin filaments. [ABSTRACT FROM AUTHOR]

1. The interaction of α with F-actin was examined with the use of the viscometric technique under various conditions. 2. α-actinin causes the gelation of F-actin when added both before and after polymerization. 3. The interaction of α-actinin with F-actin seems to be much stronger at 0° than at 21°. In the latter case it takes place only when actin is polymerized with 0.1 M KCI, but not with 1 mM MgCI2 or CaCl2. 4. Tropomyosin, when added before α-actinin, prevents the formation of F-actin gel, and when added to already formed gel, causes its disappearance. This abolishing effect of tropomyosin is much stronger at 21° than at 0°. 5. The results are discussed in view of the possible competition between tropomyosin and α-actinin for the binding to F-actin. [ABSTRACT FROM AUTHOR]

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.

Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.

1. Both TN-T and TN-I components of troponin interact with F-actin, causing its precipitation at 0.1 M KC1 and neutral pH in a form of highly ordered paracrystals, although the ability of TN-I component to precipitate of F-actin is much weaker. 2. F-actin paracrystals obtained in the presence of both TN-T and TN-I components consist of parallel arrays of F-actin filaments, although the fine structure is in each case different. 3. In the presence of tropomyosin in the proportion equal to that in muscle, less TN-T or TN-I component is needed to obtain full precipitation of F-actin. 4. Paracrystals of F-actin-tropomyosin-TN-T component and F-actin-tropomyosin-TN-I component show regular transverse striation spaced at about 380 A intervals. 5. The TN-C component of troponin solubilizes all precipitates of F-actin with TN-T or TN-I components, regardless of the presence of tropomyosin. 6. The results show that both TN-T or TN-I components can bind independently to F-actin-tropomyosin complex with the same periodicity, similar to that of the whole troponin in the living muscle.