Ewa Nowak

The origin of 30,000 dalton protein in troponin preparations

Received 17 November 197 2 1. Introduction It is already well established that the Ca2+ sensi- tivity of actomyosin is rendered by the regulatory protein system, composed of troponin and tropo- myosin [ 1,2] . Troponin appeared to consist of sever- al components which can be separated by DEAE- Sephadex chromatography [3,4] . We have recently shown [4,5] that out of three main constituents of troponin called: TN-C (mol. wt. 18,000), TN-I (mol. wt. 24,000) and TN-B (mol. wt. 39,000) (the average molecular weights and the nomenclature were taken from [5] ) two latter proteins are very susceptible to the proteolytic digestion. Thus, the other constituent of troponin corresponding to the protein of molecu- lar weight 13,000- 14,000 daltons, was found to be the product of catheptic degradation of TN-I, formed during preparation of troponin, namely during incuba- tion at pH 4.6 [4, S] . In addition, troponin prepara- tions contained usually variable amounts of another protein of molecular weight of about 30,000 daltons [4, 6-81 . We have recently found particularly large amounts of this protein in troponin preparations from slow and cardiac muscles [9] concominantly with considerable decrease of TN-B. The observation suggested that 30,000 component might derive from TN-B. This assumption obtained experimental evi- dence in the present work.

Some functional properties of nonpolymerizable and polymerizable tropomyosin.

SummaryThe binding of125I-labelled nonpolymerizable (brain or carboxypeptidase A-treated skeletal muscle) and polymerizable (intact skeletal muscle) tropomyosin to muscle F-actin was studied by ultracentrifugation under various conditions. The amount of nonpolymerizable tropomyosin bound to F-actin both in 0.1m KCl and in 7mm MgCl2 was much lower than that of the polymerizable one. In the presence of MgCl2 the amount of nonpolymerizable tropomyosin bound to F-actin approached saturation level. Under these conditions, however, the amount of skeletal muscle tropomyosin bound exceeded saturation, suggesting formation of both head-to-tail polymers and side-to-side aggregates. The latter seems to be responsible for the inhibition of acto-heavy meromyosin ATPase activity which is caused by skeletal muscle tropomyosin but not by nonpolymerizable tropomyosin.Nonpolymerizable tropomyosin can substitute for the rabbit skeletal muscle tropomyosin in the regulatory system operating in skeletal muscle. Inhibition of ATPase activity of acto-heavy meromyosin by nonpolymerizable tropomyosin in the presence of troponin and the absence of calcium ions is less than that obtained with polymerizable tropomyosin. The inhibition of ATPase activity is directly correlated with the extent of binding of nonpolymerizable tropomyosin to F-actin under the conditions of the ATPase assay.

Comparative studies of chicken gizzard and rabbit skeletal tropomyosin

Abstract 1. 1. Physicochemical and functional properties of chicken gizzard tropomyosin were compared to those of rabbit skeletal tropomysin. 2. 2. In spite of the different role tropomyosin plays in smooth and skeletal muscle, the properties and structure of gizzard and skeletal tropomyosin are very similar. 3. 3. Chicken gizzard tropomyosin has the same ability to bind to actin and troponin as the skeletal one and can fully substitute the latter in its function in the regulatory system operating in skeletal muscle.

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Gałazkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.

Troponin I and caldesmon restrict alterations in actin structure occurring on binding of myosin subfragment I

The effect of troponin I and caldesmon on phalloidin‐rhodamine‐ and 1,5‐IAEDANS‐labelled actin in skeletal muscle ghost fibers was investigated by polarized fluorescence. Both these proteins inhibited the structural alterations in the actin monomer and the increase of flexibility of actin filaments occurring on binding of myosin heads, and their effects were potentiated by tropomyosin. This immobilization of the actin filament through troponin I and caldesmon seems to originate from restriction of the relative motions of the two domains within the monomer.

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

The effect of non‐muscle tropomyosin on the interaction of filamin with F‐actin

not received F-actin Filam in Skeletal muscle tropomyosin Bovine brain tropomyosin Tropomyosin

Interaction of F-actin with troponin constituents

Abstract 1. 1.|Interaction of F-actin with troponin and its constituents has been studied. F-Actin is precipitated only by preparations of troponin which contain a high ratio of the 39 000 daltons component (TN-T) to the 19 000 daltons component (TN-C). The precipitates obtained contain most of TN-T fraction present in the original troponin preparations. Removal of the TN-T component from troponin abolishes its ability both to precipitate F-actin and to bind to this protein during sedimentation in the ultracentrifuge. Troponin causes polymerization of part of G-actin in salt-free solutions. This property is also connected with the presence of the TN-T component. 2. 2.|The isolated TN-T component also causes precipitation of F-actin in the form of paracrystals, which are dissolved by the TN-C component. All the results obtained indicate that among all constituents of troponin, the TN-T component is the protein which has a high affinity to F-actin. 3. 3.|Crude actin preparations contain a similar amount of tropomyosin-troponin impurities regardless of the temperature of extraction from acetone-dried-muscle powder. Actin extracted at 0 °C contains, however, only traces of the TN-T component. The presence of this component in actin extracted at room temperature seems to be responsible for the fact that the rest of the tropomyosin-troponin complex is tightly bound to F-actin and is hardly removed by ultracentrifugation.

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.

Polymerization of actin induced by actin-binding fragments of caldesmon

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.