Tropomyosin Binding Research Articles

A Direct Regulatory Role for Troponin T and a Dual Role for Troponin C in the Ca2+ Regulation of Muscle Contraction

Troponin (Tn), containing three subunits: Ca2+ binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca2+ regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnI.TnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnI.TnC complex, there is an activation of actomyosin ATPase that is Ca(2+)-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnId57 (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnI.TnC.TnT) and mutant (TnId57.TnC.TnT) Tn complexes demonstrated equivalent activity in the Ca2+ regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnId57 could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnId57 does not interact with TnT, these results suggest that TnT reconstitutes native Ca2+ sensitivity via direct interaction with TnC. Thus Ca2+ binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.

Cloning of Tropomodulin cDNA and Localization of Gene Transcripts during Mouse Embryogenesis

Tropomodulin (Tmod) is a tropomyosin-binding protein involved in the structuring of actin filaments. This report describes Tmod expression in distinct patterns during embryonic development in a wider variety of adult and embryonic vertebrate tissues than previously reported. Identical Tmod cDNAs were cloned from mouse brain, skeletal muscle, heart, and hematopoeitic cells. Genomic blotting demonstrates that Tmod is encoded by a single gene, which has a 1077-bp open reading frame that is highly homologous to that of the human erythrocyte. The spatial and temporal expression of the Tmod gene was examined during mouse embryogenesis using in situ hybridization. Tmod mRNA is present by 9.5 days postcoitum (p.c.) in the developing rostral somites, coincident with expression of contractile protein genes in myotomes, suggesting that Tmod may play an important role in sarcomeric thin filament organization in skeletal muscle. While the expression of Tmod mRNA in cardiac muscle is earlier than that in skeletal muscle, its appearance in the heart also coincides with the expression of genes for thin filament proteins and correlates with initial myocardial contractions at 8.0 days p.c. Tmod mRNA is not detected in developing smooth muscle of the gut, but Tmod mRNA is expressed in hematopoeitic cells in yolk sac and developing liver. The sensory ganglia and epithelia of the inner ear express Tmod mRNA as do other sensory neurons such as those in the olfactory epithelium. Expression levels in the brain are much lower prenatally than postnatally. These findings show that Tmod expression in many cell types is developmentally regulated, suggesting that the interaction of actin filaments with this tropomyosin binding protein is an important process in tissue and cell differentiation.

Isoform-specific interaction of tropomodulin with skeletal muscle and erythrocyte tropomyosins.

Tropomodulin is a tropomyosin-binding protein that localizes to the pointed end of striated muscle thin filaments and caps the pointed end of tropomyosin-actin filaments in vitro. Results from previous studies have suggested that tropomyosin-tropomodulin interactions are isoform-specific. To investigate the molecular basis for the isoform specific interactions of tropomyosin and tropomodulin, we isolated a cDNA for chicken skeletal muscle tropomodulin. The derived amino acid sequence of muscle tropomodulin is 86% identical with that of human erythrocyte tropomodulin, indicating that tropomodulins are highly conserved proteins. Multiple mRNAs seen on Northern blots of chicken skeletal muscle mRNA can be accounted for by multiple polyadenylation signals in the 3'-untranslated region of the cDNA. 125I-Labeled skeletal muscle and erythrocyte tropomyosins were assayed for binding bacterially expressed muscle tropomodulin using a solid phase binding assay. Unexpectedly, skeletal muscle and erythrocyte tropomyosins bound with similar affinities to muscle tropomodulin (Kd values approximately 0.2 microM). However, cross-competition studies using erythrocyte and skeletal muscle tropomyosins indicated that they bound different sites on tropomodulin. Competition studies using recombinant fragments of tropomodulin to map the binding domains for the two tropomyosins demonstrated that residues 6-94 contained the skeletal muscle tropomyosin binding site and residues 90-184 contained the erythrocyte tropomyosin binding site. Binding of different regions of tropomodulin to muscle and non-muscle tropomyosins may permit interaction of tropomodulin with tissue-specific components or may influence the pointed end capping activity of tropomodulin.

Tropomyosin Length and Two-stranded F-actin Flexibility in the Thin Filament

In muscle thin filaments, each tropomyosin molecule is considered to be a rope-like structure that winds along the filament in contact with seven consecutive actin monomers on the same strand of the two-stranded actin helix. Taking into account the head-to-tail overlap of the tropomyosin molecule, the effective length of this "rope" is about 405 Å, which is believed to be conserved. Tropomyosin appears to the neither extensible nor compressible in its axial direction, although it may possess much flexibility in the transverse direction. During the "maximally on" state, characterized by the presence of Ca 2+ and the strong binding between actin and myosin subfragment 1, the following conditions are thought to occur: the motion and associated flexibility of tropomyosin are reduced; the actin filament flexibility increases; a maximum number of equivalent tropomyosin binding sites on actin are concurrently saturated; and the tropomyosin molecule maintains an average thin filament radius of 38 to 40 Å. Under these potentiated conditions, the length of tropomyosin can be used to determine the limits on the underlying "cumulative angular disorder" of the actin filament with which it interacts. Our calculations show that only a small amount (∼ 1 to 3°) of this type of actin monomer rotational disorder is possible at this stage of the contractile cycle, unless the length of the tropomyosin is increased substantially between the head-to-tail joints. However, if the dominant type of F-actin rotational flexibility is between two relatively rigid actin strands (the lateral slipping/rotational offset model), all of the above actin-tropomyosin interactions can be completely and easily accommodated. We also discuss the implications of an interdomain hinge in G-actin and the possibility that there may be fewer than seven equivalent sites on actin that are saturated by tropomyosin concurrently.

Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon

We expressed the following fragments of chicken gizzard caldesmon in the pMW 172/BL21 (DE3) system at 0.4-2.2 mg of pure protein/liter of culture: full-length smooth muscle caldesmon (CDh) (amino acids 1-756), nonmuscle caldesmon (CDl), amino acids 1-128 (N128), 1-578 (N578), 230-419, 606-756 (606C), and 658-756 (658C). CDh bound tropomyosin with a Kd of 1.5 microM; N578, 230-419, and 606C bound with affinities at least 2-5 fold lower; N128 bound weakly; and 658C did not bind. Only N128 and N578 bound to smooth muscle myosin, both about 10-fold weaker than CDh and CDl. Only 606C and 658C bound to actin-tropomyosin with affinities CDh = 606C > 658C. The binding to actin-tropomyosin was biphasic, whereas the binding to actin was monophasic, corresponding to the weak binding component found in the presence of tropomyosin. Calmodulin bound only to the C-terminal fragments with the same affinity as CDh. CDh, 606C, and 658C inhibited actin-tropomyosin-activated myosin ATPase, with maximal inhibition correlated with 1 caldesmon bound/14 actins, and inhibition was reversed by Ca(2+)-calmodulin. Thus, the actomyosin ATPase regulatory function, calmodulin binding, and most actin binding is located within the C-terminal 99 amino acids, whereas tropomyosin binding is distributed throughout the rest of the molecule.

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.

Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.

The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.

Yeast actin is relatively well behaved

Actin from yeast has been reported previously to have unusual polymerization properties. Here we report a simple sensitive spot assay for actin and use it to develop a high-yield procedure for the purification of actin from the yeast Saccharomyces cerevisiae. The polymerization properties of purified yeast actin are quantitatively similar to all other characterized actins. We have characterized this actin with respect to its ability to interact with yeast profilin and tropomyosin, the only yeast actin-binding proteins so far purified and characterized. Yeast profilin can sequester yeast actin monomers and thereby reduce the ability of yeast actin to polymerize, whereas it has little effect on the degree of polymerization of rabbit skeletal muscle actin. By contrast, there is no apparent difference between the binding of yeast or smooth muscle tropomyosin to yeast or rabbit skeletal muscle actin. The availability of purified yeast actin should facilitate a detailed examination of its interaction with recently discovered yeast actin-binding proteins. Greer and Schekman (1982) [Greer, C. & Schekman, R. (1982), Mol. Cell Biol. 2, 1279-1286] reported that an intrinsic property of yeast actin is a Ca2+ dependent increase in critical concentration with the formation of 15-50-nm particles. Our purified actin does not have this property. By modifying the purification protocol, we can obtain a preparation having a Ca(2+)-dependent change in polymerization properties. The Ca(2+)-dependent effect results in a slower polymerization rate as well as the formation of shorter filaments. Since this effect could be mediated by a protein present at a very low stoichiometry to actin, and we do not see any contaminating peptides, we have not pursued this effect further. We suggest that the Ca(2+)-dependent properties of the Greer and Schekman preparation are most likely due to a minor contaminant.

Site-specific amino-terminal mutants of yeast-expressed β-actin Characterization of the interaction with myosin and tropomyosin

Neutral or charge-shifting, mutagenesis of β-actin at positions 3 and 4 strongly influenced the actomyosin interaction under non-rigor conditions. The polymerization behaviour and tropomyosin binding properties on the other hand remained unaffected.

Tropomodulin binding to tropomyosins. Isoform-specific differences in affinity and stoichiometry.

Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471-482]. We have characterized the interaction of erythroid and non-erythroid tropomyosins with tropomodulin by non-denaturing gel electrophoresis and by solid-phase binding assays using 125I-tropomyosin. Non-denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non-erythrocyte tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd = 14 nM-1 and 5 nM-1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross-linking data which indicate that tropomodulin can self-associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N-terminal or C-terminal sequences characteristic of the different tropomyosin isoforms. Isoform-specific interactions of tropomodulin with tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions.

Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.

Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.

Rate and mechanism of the assembly of tropomyosin with actin filaments

The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM MgCl2, 100 mM KCl, pH 7.5, 25 degrees C) and at the protein concentrations used (2.5-24 microM actin, 0.2-3.4 microM tropomyosin) the half-life time of assembly of tropomyosin with actin filaments was found to be less than 1 s. The results were analyzed quantitatively by a model in which tropomyosin initially binds to isolated sites. Further tropomyosin molecules bind contiguously to bound tropomyosin along the actin filaments. Good agreement between the experimental and theoretical time course of assembly was obtained by assuming a fast preequilibrium between free and isolatedly bound tropomyosin.

Tropomyosin is co-localized with the actin filaments of the scolopale in insect sensilla

Immuno-electron microscopy confirms that the scolopale, a characteristically prominent cytoskeletal element of insect scolopidia, is composed mainly of actin filaments. Immunohistochemistry reveals that these filaments are co-localized with tropomyosin. Myosin S1-decoration shows that their polarity is unidirectional. Antibodies to α-actinin do not bind within the scolopale. The association of these actin filaments with tropomyosin in the absence of myosin, together with their uniform polarity, strongly suggests that, in the scolopale, they have a stabilizing rather than contractile function. Filament elasticity would appear to be important for stimulation. The degree of elasticity may well be governed by the extent of tropomyosin binding.

Interaction of maleimidobenzoyl actin with myosin subfragment 1 and tropomyosin-troponin

A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl (pH 7.6). The resulting polymer (MBS-P-actin) is highly potentiated so that it activates the Mg(2+)-ATPase of S1 more strongly than native F-actin. The affinity of MBS-P-actin for S1 in the presence of ATP (KATPase) is about four times higher than that of native F-actin, although the maximum velocity at infinite actin concentration (Vmax) is almost the same. This high activation is not due to a cross-linking between MBS-P-actin and the S1 heavy chain, since no substantial amount of cross-linking was observed in SDS gel electrophoresis. Direct binding studies and ATPase measurements showed that the modification of actin with MBS impairs the binding of tropomyosin. Tropomyosin binding can be improved considerably by the addition of troponin. However, the regulation mechanism of the acto-S1 ATPase activity by troponin-tropomyosin is damaged. The addition of troponin-tropomyosin reduces the S1 ATPase activation by MBS-P-actin to the same level as that of native F-actin in 30 mM KCl/2.5 mM ATP/2 mM MgCl2, but there is no difference in the ATPase activation in the presence and absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calponin and the composition of smooth muscle thin filaments

Recent work suggests that caldesmon and calponin, two actin-binding proteins found in smooth muscle, compete with each other for interaction with F-actin, implying that they may not be subunits of the same complex in vivo. Rather than associating with the same thin filaments in vivo, the two proteins actually may be localized on separate filaments possibly performing distinct functions. It is well-established that actomyosin ATPase and consequently tension in vertebrate smooth muscle are dependent on Ca2+-calmodulin-activated phosphorylation of myosin light chain subunits (reviewed in Kamm & Stull, 1985). The possibility of additional thin filament-linked modulation, raised by the pioneering work of Driska and Hartshome (1975) and Ebashi and colleagues (1977) and detailed extensively by Marston and colleagues (Marston et al., 1980; Marston & Smith, 1984), also has received much attention. This possibility became particularly attractive after Sobue and colleagues (1981) first isolated and characterized the protein caldesmon, which now is known to be localized exclusively on thin filaments (Marston & Lehman, 1985) and capable of modulating actomyosin ATPase in vitro (Sobue eta]., 1981; Ngai & Walsh, I984; Dabrowska et al., 1985; Marston & Lehman, I985; Chacko et al., I987). More recently, a second putative thin filament-linked regulatory protein, calponin, has been characterized by Takahashi and colleagues (1986, 1988, 1990), and their work on this 34-35 kDa molecule has stimulated further interest in the role played by thin filament-binding proteins in the contractility of smooth muscle. In these studies, antibody crossreactivity and molecular cloning demonstrated that regions of calponin have an amino acid sequence homologous to one of the tropomyosin binding domains of skeletal muscle troponinT, suggesting strongly that calponin is a true thin filament binding protein (Takahashi et al., 1988, 1990). Calponin also shares extensive sequence homology with the very abundant and modestly-named smooth muscle protein, SM 22, which as yet has no known function or binding affinities (Vancompernolle et al., 1990; Takahashi et al., 1990; Lees-Miller et al., 1987; Pearlstone et al., 1987). Calponin, like caldesmon, binds to actin with high affinity and also binds calmodulin (Lehman & Kaminer, 1984; Takahashi et al., 1986; Lehman, 1989; Winder & Walsh, 1990; Sutherland eta]., I990). However, with both caldesmon and calponin potentially interacting with actin, models of smooth muscle thin filaments seem to be becoming somewhat congested. Clearly a more complete understanding of the composition, assembly, and distribution of thin filaments in smooth muscle is required to clarify the roles played by these thin filament-binding proteins. A number of studies analyzing reconstituted and intact thin filaments have begun to address these questions and provide direction for future investigation. Experiments in the laboratories of Shirinsky and Dabrowska may have yielded a key piece of evidence pertinent to our understanding of how caldesmon and calponin interact with actin. In their studies, F-actin complexed with purified calponin was exposed to increasing concentrations of caldesmon; this resulted in binding of the caldesmon and displacement of bound calponin. Analogously, they also found that calponin was able to displace caldesmon bound to actin filaments (Makuch eta]., 1991). Hence, caldesmon and calponin compete for actin binding sites on thin filaments and apparently do not form a complex with each other, as do the subunits of troponin on skeletal muscle thin filaments. This would explain why, in some preparations, calponin seems to interfere with caldesmon inhibition of actomyosin ATPase (S. B. Marston, personal communication) and, in others, caldesmon with calponin inhibition (Sutherland eta]., 1990). Surprisingly, the presence of tropomyosin does not seem to influence calponin inhibition of ATPase (Makuch eta]., 1990; Winder & Walsh, 1990), and its role in calponin function and in calponin binding on thin filaments remains undefined. Even though calponin and caldesmon both bind to actin, it seems unlikely that they are randomly or even sequentially arranged on individual thin filaments in vivo. Caldesmon, at least, is regularly and periodically distributed on relatively intact, isolated, thin filaments in

Binding Mode of Cytochalasin B to F-Actin Is Altered by Lateral Binding of Regulatory Proteins

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin.

Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues.

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation.

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.

Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon

The fraction of polyclonal caldesmon antibodies cross-reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in M r 77 kDa non-muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.