Absence Of Tropomyosin Research Articles

CacyBP/SIP as a novel modulator of the thin filament

The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin–tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

LRR Domain of Tropomodulin is Responsible for Targeting it to the Pointed End of the Actin Filament

Tropomodulin, a tropomyosin-dependent actin filament capping protein, consists of two structurally and functionally different domains. Tropomodulin lacking its C-terminal domain can cap actin filaments with an affinity close to the full-length protein in vitro. To investigate the functional properties of the C-terminal domain in live cells, truncated GFP-tagged tropomodulin was expressed in rat cardiac myocytes. Three fragments were analyzed: Tmod1(1-159) that lacks the entire C-terminal domain, Tmod1(1-320) and Tmod1(1-349). GFP-Tmod1(1-159) did not assemble well at the pointed ends of the filaments (∼80% of the cells demonstrated a diffuse distribution, while ∼20% showed faint, inconsistent assembly). Together, the in vitro and live cell studies indicate that the C-terminal domain is not required for capping actin filaments but is important for specifically targeting tropomodulin to the pointed ends of the actin filaments in sarcomeres. In the cells where GFP-Tmod1(1-320) was expressed most (∼70%) of the assembly was faint and inconsistent. Tmod1(1-320) lacks the 39 C-terminal residues that include both the C-terminal helix that is not a part of LRR fold, and the tropomyosin independent actin filament capping site. The precise location of the tropomyosin independent actin filament capping site is not known, although removal of 15 residues from the C-terminus destroys the actin filament capping ability of Tmod1 in the absence of tropomyosin. GFP-Tmod1(1-349) is missing the ten C-terminal residues which discriminates Tmod1 from other tropomodulin isoforms; this fragment consistently assembled at the thin filament pointed ends, comparable to the cells expressing wild type GFP-Tmod1. Based on these data we suggest that both the LRR fold (residues 160-320) and residues 321-349 are important for regulating tropomodulin's pointed end capping activity though the specific role each of these regions play in this phenomenon may be different.

Troponin Is a Potential Regulator for Actomyosin Interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.

Fesselin binds to actin and myosin and inhibits actin-activated ATPase activity

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca(++)-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca(++)-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with muM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions.

The Regulatory Effects of Tropomyosin and Troponin-I on the Interaction of Myosin Loop Regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088-5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac beta-myosin, presumed to be sites of interaction with F-actin (residues 554-584, 622-646, and 633-660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134-147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.

Identification and Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.

Influence of ionic strength, actin state, and caldesmon construct size on the number of actin monomers in a caldesmon binding site.

There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of caldesmon concentrations, the type of caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that caldesmon binds tightly to 2-3 actin monomers. Additional parts of caldesmon bind less tightly to actin, causing caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.

Cooperative inhibition of actin filaments in the absence of tropomyosin.

We measured the inhibition of actin activated myosin subfragment-1 MgATPase activity in a solution containing no added KCl (5 mM PIPES.K2 (pH 7.1), 2.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, 5 mM MgATP). Maximal inhibition was observed with substoichiometric concentrations of caldesmon, caldesmon domain 4, troponin and troponin I. In six experiments using different preparations of actin, S-1 and caldesmon 50% inhibition required 0.09 +/- 0.01 (sem) caldesmon added per actin. This compares with 0.66 +/- 0.32 (sem, n = 5) caldesmon per actin for 50% inhibition in the presence of 60 mM KCl. With caldesmon domain 4, 50% inhibition was achieved with 0.17 +/- 0.08 (n = 11) domain 4 added per actin. We measured the amount of caldesmon bound at the same time as inhibition. Complete inhibition of actin activated ATPase needed only one caldesmon bound per 5.0 +/- 0.5 (sem, n = 5) actin monomers or one caldesmon domain 4 bound per 3.9 +/- 0.6 (sem, n = 3) actin monomers at zero KCl. We conclude that under these conditions inhibition of actin is cooperative despite the absence of tropomyosin. We measured the effect of caldesmon inhibition upon S-1 binding to actin. S-1.ADP.Pi (weak binding) was not affected by caldesmon concentrations giving 80% inhibition, however S-1.ADP (strong binding) was highly cooperative, being very weak at <0.3 microM but indistinguishable from uninhibited actin at >2 microM S-1.ADP. We conclude that actin can exist in two activity states corresponding to the 'on' and 'off' states of actin-tropomyosin and inhibitory proteins function as allosteric-cooperative inhibitors of actin. The implications of these findings for the role of tropomyosin in thin filament regulation are discussed.

Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge.

Rapid polymerization of a network of short, branched actin filaments takes place at the leading edge of migrating cells, a compartment enriched in activators of actin polymerization such as the Arp2/3 complex and cofilin. Actin filaments elsewhere in the cell are long and unbranched. Results reported here show that the presence or absence of tropomyosin in these different actin-containing regions helps establish functionally distinct actin-containing compartments in the cell. Tropomyosin, an inhibitor of the Arp2/3 complex and cofilin function, was localized in relation to actin filaments, the Arp2/3 complex, and free barbed ends of actin filaments in MTLn3 cells, which rapidly extend flat lamellipodia following EGF stimulation. All tropomyosin isoforms examined using indirect immunofluorescence were relatively absent from the dynamic leading edge compartment, but did colocalize with actin structures deeper in the lamellipodium and in stress fibers. An in vitro light microscopy assay revealed that tropomyosin protects actin filaments from cofilin severing. The results suggest that tropomyosin-free actin filaments under the membrane can participate in rapid, dynamic processes that depend on interactions between the activities of the Arp2/3 complex and ADF/cofilin that tropomyosin inhibits elsewhere in the cell.

Tropomyosin keeps actin tough

![Graphic][1] Actin (green) and ADF/cofilin (red) are disorganized in the absence of tropomyosin (bottom). The actin cytoskeleton is a highly adaptable framework. In some structures actin polymerizes and depolymerizes rapidly to facilitate quick movement, whereas in others actin

Tropomodulin Increases the Critical Concentration of Barbed End-capped Actin Filaments by Converting ADP·Pi-actin to ADP-actin at All Pointed Filament Ends

The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration.

Characterization of the functional properties of smooth muscle caldesmon domain 4a: evidence for an independent inhibitory actin-tropomyosin binding domain.

Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715-793) and one is in domain 4a (amino acids 636-714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a: H10 [622-708], H12 [506-708] and H13 [622-726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin-tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b.

The rate of annealing of actin tropomyosin filaments depends strongly on the length of the filaments

Actin tropomyosin filaments were sheared to produce short filaments. Following incubation for 0 to 10000 s annealing of the filaments was assayed by determination of the rate of polymerization of monomeric actin onto the filament ends. The rate of decrease of the concentration of filament ends was found to be proportional to its fourth power. In contrast, the rate of end-to-end association of actin filaments in the absence of tropomyosin was proportional to the square of the concentration of filament ends. The strong dependence on the filament length of the rate of annealing of actin tropomyosin filaments was interpreted by the model of Hill (Biophys. J., 44, 285–288 (1983)) who pointed out that the rate constant of end-to-end association of long rod-like filaments is expected to depend on the length of the filaments for sterical conditions.

In Vitro Motility Analysis of Smooth Muscle Caldesmon Control of Actin-Tropomyosin Filament Movement

We have used the in vitro motility assay to investigate the effect of caldesmon on the movement of actin-tropomyosin filaments over thiophosphorylated smooth muscle myosin and skeletal muscle heavy meromyosin. Using either motor, incorporation of up to 8 nM caldesmon inhibited filament movement by decreasing the proportion of filaments motile from > 85% to < 30%. There was a minimal effect on filament attachment and a modest decrease in motile filament velocity in this concentration range. The reduction in the proportion of filaments motile could be completely reversed by incorporation of an excess of calmodulin at pCa 4.5. The expressed C-terminal fragment, 606C, which retains caldesmon's inhibitory capacity but does not bind to myosin, decreased the proportion of filaments motile but had no effect on velocity. We conclude that the velocity reduction by whole caldesmon is due to actin-myosin cross-linking. A significant decrease in filament attachment was observed when caldesmon was added to an excess over actin (> 10 nM). In the absence of tropomyosin, addition of an excess of caldesmon caused a similar decrease in the filament density, but there was no effect on the proportion of filaments that were motile. Our results demonstrate that caldesmon can switch actin-tropomyosin from motile to non-motile states without controlling velocity of movement or weak binding affinity and show the inhibitory action of caldesmon in the motility assay to be functionally indistinguishable from that reported for troponin.

The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.

Tropomodulin caps the pointed ends of actin filaments.

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

Inhibition of Actin Stimulation of Skeletal Muscle (A1)S-1 ATPase Activity by Caldesmon

We have previously shown that caldesmon inhibits the actin-activated ATPase activity of myosin subfragments in parallel with inhibition of myosin subfragment · ATP binding to actin (M. E. Hemric, and J. M. Chalovich, 1988, J. Biol. Chem. 263, 1878-1885; L. Velaz, R. H. Ingraham, and J. M. Chalovich, 1990, J. Biol. Chem. 265, 2929-2934). From these data, we suggested that caldesmon is a competitive inhibitor of binding of myosin subfragment-1 to actin. To confirm this result, we now show the effect of caldesmon on the steady-state parameters of ATP hydrolysis by (A1)S-1 at increasing actin concentrations. Low ionic strength conditions were used to maximize the interaction between (AL)S-1 and actin. In both the presence and absence of smooth muscle tropomyosin, caldesmon caused a twofold decrease in the k cat and more than a 12-fold change in the K ATPase. Therefore, competition of binding of myosin to actin by caldesmon contributes to the reduction in ATPase activity in both the presence and the absence of tropomyosin.

The interaction of caldesmon with the COOH terminus of actin.

Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin.

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.