The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.     

Caldesmon reduces the apparent rate of binding of myosin S1 to actin-tropomyosin

Equilibrium measurements of the rate of binding of caldesmon and myosin S1 to actin-tropomyosin from different laboratories have yielded different results and have led to different models of caldesmon function. An alternate approach to answering these questions is to study the kinetics of binding of both caldesmon and S1 to actin. We observed that caldesmon decreased the rate of binding of S1 to actin in a concentration-dependent manner. The inhibition of the rate of S1 binding was enhanced by tropomyosin, but the effect of tropomyosin on the binding was small. Premixing actin with S1 reduced the amplitude (extent) of caldesmon binding in proportion to the fraction of actin that contained bound S1, but the rate of binding of caldesmon to free sites was not greatly altered. No evidence for a stable caldesmon-actin-tropomyosin-S1 complex was observed, although S1 did apparently bind to gaps between caldesmon molecules. These results indicate that experiments involving caldesmon, actin, tropomyosin, and myosin are inherently complex. When the concentration of either S1 or caldesmon is varied, the amount of the other component bound to actin-tropomyosin cannot be assumed to remain fixed. The results are not readily explained by a mechanism in which caldesmon acts only by stabilizing an inactive state of actin-tropomyosin. The results support regulatory mechanisms that involve changes in the actin-S1 interaction.     

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin

Application of the myosin competition test (Lehman, W., and Szent-Gy?rgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.     

Caldesmon-binding sites on tropomyosin

The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).     

Bundling of actin filaments by aorta caldesmon is not related to its regulatory function

Ca2+-sensitive thin filaments from vascular smooth muscle were disassembled into their constituent proteins, actin, tropomyosin and caldesmon. Caldesmon bound to both actin and to actin-tropomyosin and inhibited actin-tropomyosin activation of skeletal muscle myosin MgATPase. It also promoted the aggregation of actin or actin-tropomyosin into parallel aligned bundles. Quantitative electron microscopy measurements showed that with 1.1 microM actin-tropomyosin, 1.6 +/- 0.5% (n = 3) of the filaments were in bundles. At 0.073 microM, caldesmon inhibited MgATPase activity by 50%, whereas bundling was 3.0 +/- 1.3% (n = 4). At 0.37 microM caldesmon, MgATPase inhibition was 83% while 28.1 +/- 6.9% (n = 4) of filaments were in bundles. Experiments at 4.4 microM in which MgATPase and bundling were measured in the same samples gave similar results. Small bundles of 2-3 filaments showed the most frequent occurrence at 1.1 microM actin. At 4.4 microM actin the most common bundle size was 3-5 filaments, with the occasional occurrence of large bundles consisting of up to 120 filaments. The incidence of bundling was the same in the presence and absence of tropomyosin. Thus caldesmon can induce the formation of actin bundles but this property bears no relationship to its inhibition of MgATPase activity.     

The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments

Chicken gizzard caldesmon causes up to 40% inhibition of Mg2+-ATPase activity of rabbit skeletal muscle actomyosin. In the presence of chicken gizzard tropomyosin this inhibition is significantly increased, reaching a maximum (around 80%) at a molar ratio of caldesmon to actin monomer of 1 to 10-13. The inhibition of actomyosin ATPase takes place over a wide pH range (from 6.0 to 8.0) but is decreased with an increase in KCl and MgCl2 concentrations. Caldesmon, in the range of caldesmon/ actin ratios within which it inhibits actomyosin ATPase, forms bundles of parallelly aligned actin filaments. Calmodulin in the presence of Ca2+ dissociates these bundles and restrains the inhibition of actomyosin ATPase, provided that it is used at a high molar excess over caldesmon.     

Caldesmon, calmodulin and tropomyosin interactions

Binary complex interactions between caldesmon and tropomyosin, and calmodulin and tropomyosin, and ternary complex interaction involving the three proteins were studied using viscosity, electron microscopy, fluorescence and affinity chromatography techniques. In 10 mM NaCl, caldesmon decreased the viscosity of chicken gizzard tropomyosin by 7-8 fold with a concomitant increase in turbidity (A330nm). Electron micrographs showed spindle-shaped particles in the tropomyosin-caldesmon samples. These results suggest side-by-side aggregation of tropomyosin polymers induced by caldesmon. Binding studies in 10 mM NaCl between caldesmon and chicken gizzard tropomyosin labelled with the fluorescent probe N-(1-anilinonaphthyl-4)maleimide (ANM) gave association constants from 5.3.10(6) to 7.9.10(6) M-1 and stoichiometry from 1.0 to 1.4 tropomyosin per caldesmon. Similar binding was observed for rabbit cardiac tropomyosin and caldesmon. Removal of 18 and 11 residues from the COOH ends of the gizzard and cardiac tropomyosin by carboxypeptidase A, respectively, had no significant effect on their binding to caldesmon. In the presence of Ca2+, chicken gizzard tropomyosin bound to a calmodulin-Sepharose-4B column and was eluted with a salt concentration of 140 mM. This interaction was weakened in the absence of Ca2+, and the bound tropomyosin was eluted by 65 mM KCl. ANM-labelled tropomyosin bound calmodulin in the presence of Ca2+ with a binding constant of 3.5.10(6) M-1 and a binding stoichiometry of 1 to 1.4 tropomyosin per calmodulin. In 10 mM NaCl, calmodulin reduced the specific viscosity of chicken gizzard tropomyosin in the presence of Ca2+ by 5 fold, while a 1.5-fold reduction in viscosity was observed in the absence of Ca2+. In either case, no significant increase in turbidity was observed suggesting that calmodulin reduced head-to-tail polymerization of tropomyosin. The interaction of caldesmon with the calmodulin-ANM-tropomyosin complex in the presence and absence of Ca2+ was also examined. The result is consistent with a model that in the absence of Ca2+, calmodulin binds weakly to either caldesmon or tropomyosin and has little effect on the tropomyosin-caldesmon interaction; whereas, Ca2(+)-calmodulin interacts with caldesmon and reduces its affinity to tropomyosin.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin

The binding of 125I- and 14C-caldesmon to actin and actin-tropomyosin was studied using a cosedimentation technique and was analyzed by the method of McGhee and von Hippel [1974) J. Mol. Biol. 86, 469-489) for the binding of large ligands to a homogeneous lattice. The binding was adequately described by a single class of binding sites with a stoichiometry between 1:7 and 1:10. The binding exhibited a small degree of positive cooperativity (omega = 5-6) which was the same in the presence and absence of tropomyosin. The association constant for the binding of caldesmon to an isolated binding site was enhanced, from about 6 X 10(5) to about 1.4 X 10(6) M-1, by the presence of smooth muscle tropomyosin. Caldesmon inhibited the actin-activated ATPase activity of skeletal myosin subfragment 1 in both the absence and presence of tropomyosin. Maximum inhibition of ATPase activity occurred when one caldesmon molecule bound to seven actin monomers. A greater degree of inhibition was observed in the presence of tropomyosin than in the absence. This greater inhibition cannot be explained totally by the increased strength of binding of caldesmon to actin in the presence of tropomyosin. Finally, Ca2+-calmodulin completely reversed the binding of caldesmon to actin.     

Effect of muscle and non-muscle tropomyosins in reconstituted skeletal muscle actomyosin

Smooth and non-muscle tropomyosins were found to produce a 2-3-fold Ca-insensitive stimulation of the ATPase activity of reconstituted skeletal muscles actomyosin at normal MgATP concentrations and physiological ratios of myosin to actin. Under the same conditions skeletal muscles tropomyosin had no effect. Similar effects of these three tropomyosins were observed for the low myosin/F-actin ratios necessary for kinetic measurements. Since it could be established that this actomyosin system, with or without tropomyosin, obeyed Michaelian kinetics, the tropomyosin effects could be interpreted in terms of their influence on maximal turnover (V) or on the affinity of myosin for actin (Kapp). Accordingly, gizzard tropomyosin had practically no effect on the affinity and reduced only slightly the value of V, compared to pure actin. In contrast to gizzard tropomyosin, brain tropomyosin produced an approximately twofold increase in both Kapp and V; i.e. it increased the turnover rate but decreased the affinity. It is apparent from the data that brain tropomyosin acts as an uncompetitive activator with respect to pure actin, while having the same V as the actin plus gizzard tropomyosin complex. Further studies on these tropomyosins show that only skeletal and smooth muscle tropomyosin have similar functional properties with respect to troponin inhibition and the activation of the ATPase at low ATP concentrations. It is suggested that the noted increases in V by tropomyosin are caused by the acceleration of the dissociation of the myosin head from actin at the end point of the cross bridge movement.     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.     

Cloning and expression of a smooth muscle caldesmon

Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data.