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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]

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.

The relaxation rate enhancements of the 23Na nuclei for NaHCO3 solutions of calmodulin and its tryptic peptides TR-1 and TR-2 indicate true binding of Na+ ions to these biomolecules. With both TR-1 and TR-2, Na+ binding occurs in competition with Ca2+ and Mg2+ binding: log KNa approximately equal to 2, log KMg apoproximately equal to 4, log KCa approximately equal to 6 for TR-1; and log KNa approximately equal to 2, log KMg approximately equal to 3, log KCa approximately equal to 5 for TR-2. All the binding constants are systemically greater for binding to TR-1, as compared to TR-2. There is also an increase in KNa for TR-1 of calmodulin as compared to the homologous tryptic fragment of troponin C. The increased binding is identified tentatively with site I of calmodulin. The binding constants KNa, KCa and KMg of calmodulin appear to be finely tuned to the intracellular concentrations of these cations.

Exchangeability of actin-bound ADP and calcium in actomyosin at low ionic strength has been studied using F-actin labelled with [14C]ADP or 45Ca and measuring release of radioactivity into solution. Low-speed centrifugation, ultracentrifugation and ultrafiltration were used to separate protein from the medium. Comparison of the results obtained with these three separation procedures has revealed that the release of [14C]ADP and 45Ca into the medium in the presence of millimolar concentrations of MgATP is largely due to the release under these conditions of actin itself retaining its bound ADP and calcium. The real exchange of the bound nucleotide and calcium, even under the most favourable conditions, was in our experiments limited to about 20%. Detailed examination of the dependence of both the release of actin and the exchange of actin-bound ADP and calcium on the free divalent cations present, the kind and concentration of the added nucleotide, and temperature of incubation indicates that there is no correlation between the exchange and superprecipitation of actomyosin. The results presented support the view that the limited enhancement by myosin of the exchange of nucleotide and cation bound to actin under certain conditions results from accidental disruption of bonds between actin monomers due to a mechanical stress exerted on actin filaments upon their interaction with myosin filaments.

Two tryptic fragments of the Ca2+ -binding protein calmodulin have been studied by high-resolution 1H NMR. TR1C (residues 1 - 77) spans the first two domains of the protein and TR2C (residues 78 - 148) spans the second two domains. The spectra indicate that each of the two-domain peptides assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modelled after the two EF-hand Ca2+-binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+-binding protein.

The relaxation rate enhancements of the 23Na nuclei for NaHCO3 solutions of troponin C and its tryptic peptides TR-1 and TR-2 indicate true binding of Na+ ions to these biomolecules. The low-affinity sites I and II of TR-1 and troponin C are the sites of competitive Na+/Ca2+ binding, below one calcium ion per molecule, with log KNa approximately 2. At low calcium content Na+ ions bind to TR-2 and to troponin C non-competitively with Ca2+ ions; binding of Ca2+ ions to the high-affinity sites III and IV allosterically affects the binding of the Na+ ions: even when sites I and II, located on TR-1 or sites I, II, III, IV of troponin C, are saturated with Ca2+ ions, Na+ ions continue to bind weakly at secondary binding sites.