Glutamyl-gamma-boronate inhibitors of bacterial Glu-tRNA(Gln) amidotransferase.

Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.

Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase.

Organisms lacking Gln-tRNA synthetase produce Gln-tRNA(Gln) from misacylated Glu-tRNA(Gln) through the transamidation activity of Glu-tRNA(Gln) amidotransferase (Glu-AdT). Glu-AdT hydrolyzes Gln to Glu and NH(3), using the latter product to transamidate Glu-tRNA(Gln) in concert with ATP hydrolysis. In the absence of the amido acceptor, Glu-tRNA(Gln), the enzyme has basal glutaminase activity that is unaffected by ATP. However, Glu-tRNA(Gln) activates the glutaminase activity of the enzyme about 10-fold; addition of ATP elicits a further 7-fold increase. These enhanced activities mainly result from increases in k(cat) without significant effects on the K(m) for Gln. To determine if ATP binding is sufficient to induce full activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependent glutaminase activity. Despite their binding to Glu-AdT, none of the ATP analogues induced glutaminase activation except ATP-gammaS, which stimulates glutaminase activity to the same level as ATP, but without formation of Gln-tRNA(Gln). ATP-gammaS hydrolysis by Glu-AdT is very low in the absence or presence of Glu-tRNA(Gln) and Gln. In contrast, Glu-tRNA(Gln) stimulates basal ATP hydrolysis slightly, but full activation of ATP hydrolysis requires both Gln and Glu-tRNA(Gln). Simultaneous monitoring of ATP or ATP-gammaS hydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities in the presence of ATP, with all three activities waning in concert when Glu-tRNA(Gln) levels become exhausted. ATP-gammaS stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitant transamidase activity and with a very low level of ATP-gammaS hydrolysis. This uncoupling between ATP-gammaS hydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-gammaS, together with Glu-tRNA(Gln), results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gammaS hydrolysis.

Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes.

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains.

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.

Kinetic effects due to nonspecific substrate-inhibitor interactions in enzymatic reactions.

Nonspecific protein binding is a commonly encountered problem with synthetic molecules designed as enzyme inhibitors. When the substrate for the enzymatic reaction is itself a protein, such nonspecific protein binding can also occur. Here, we demonstrate that this phenomenon can have a dramatic effect on the steady-state kinetic evaluation of such inhibitors.

Identification of a novel inhibitor of mitogen-activated protein kinase kinase.

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Competitive inhibition of MAP kinase activation by a peptide representing the alpha C helix of ERK.

On the basis of the crystal structure of the MEK substrate ERK, we have synthesized a 15 amino acid peptide representing the alpha C helix of human ERK1. We find this peptide to be an inhibitor of ERK phosphorylation by its upstream activator MEK. Circular dichroic spectroscopy indicates that the peptide has little secondary structure in aqueous buffer, but can readily adopt an alpha-helical structure in aprotic solvent. Steady-state kinetic analysis indicates that the peptide serves as a competitive inhibitor of ERK binding to MEK, with a dissociation constant, Ki, of 0.84 microM. Together with ATP-competitive inhibitors of MEK, we have used this peptide to define the kinetic mechanism of MEK catalysis. These studies reveal that MEK operates through a bi-bi random-ordered sequential mechanism. The synthetic peptide inhibits also the phosphorylation of p38 and ERK by the upstream activator MKK3, but is at least 3-fold less potent as an inhibitor of SEK activation of JNK1. Interestingly, the peptide also showed some ability to inhibit ERK-mediated phosphorylation of myelin basic protein, but was inactive as an inhibitor of the unrelated kinases Raf, Abl, and PKA. These results imply that the alpha C helix is an important locus of interaction for the formation of a MEK-ERK complex. The alpha C helix cannot, however, be the sole determinant of activator selectivity among the MAP kinases. Molecules designed to target the alpha C helix binding pocket of MAP kinase activators may provide a novel means of inhibiting these signal transducers.

Modulation of actin conformation and inhibition of actin filament velocity by calponin.

Calponin, an actin/calmodulin-binding protein present in smooth muscle thin filaments, modulates the actin-myosin interaction and actomyosin ATPase activity of smooth muscle myosin II. Binding of myosin heads to actin under conditions that produce weak or strong binding induces conformational changes in actin. Polarized fluorimetric measurements of rhodamine-phalloidin complex and 1,5-IAEDANS specifically linked to actin in myosin-free muscle fibers (ghost fibers) and to Cys-707 in myosin head, respectively, revealed conformational changes, as determined from the changes in orientation and mobility of fluorescent probes, upon addition of calponin to ghost fibers. The effect of calponin on conformational changes produced upon binding of phosphorylated or dephosphorylated heavy meromyosin (HMM) was also determined. Subfragment-1 preparation modified with NEM (NEM-S1) or pPDM (pPDM-S1) were used as models of strong and weak binding, respectively. Calponin changed both the orientation of fluorophores on the actin and the flexibility of the actin filaments, as determined from the angle between an actin filament and the fiber axis. Changes in the flexibility of actin filaments and the orientation of fluorophores produced by phosphorylated smooth muscle HMM were similar to those seen with NEM-S1, which formed a strong-binding association with actin and caused the transition of actin monomers to the "on" state; calponin markedly inhibited this effect. In contrast, pPDM-S1 and dephosphorylated HMM induced weak binding and the transition of actin monomers to the "of" state, and these effects were enhanced by calponin. Furthermore, calponin decreased the velocity of actin filament movement over skeletal muscle myosin O gamma phosphorylated smooth muscle myosin heads in an in vitro motility assay. These results suggest that calponin induces modulation of smooth muscle contraction by inhibiting the force-producing (strong-binding) state of cross-bridges and involves changes in actin conformation.

Characterization of the functional domains on the C-terminal region of caldesmon using full-length and mutant caldesmon molecules.

A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690-717, 658-689, and 628-657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.

[The effect of a caldesmon fragment with a mol. weight of 38 kDa and calponin on the capacity of actin to form a "strong" form of binding with myosin heads]. / Vliianie fragmenta kal'desmona s mol. massoi 38 kDa i kal'ponina na sposobnsot' aktina obrazovyvat' s golovkami miozina "sil'nuiu" formu sviazyvaniia.

Effect of calponin and 38 kD actin-binding proteolytic fragment of caldesmon on actin structure alterations, initiated by decoration of thin filaments by N-ethylmaleimide-modified skeletal myosin subfragment-1 (NEM-S1) and by phosphorylated smooth heavy meromyosin (pHMM), has been studied by polarized fluorimetry. F-actin of myosin-free ghost fiber was labeled with fluorescent probe fluoroscein-5-maleimide. Both the actin-binding regulatory proteins have been demonstrated to inhibit conformational changes of actin typical for the "strong" binding of myosin head to actin. Tropomyosin weakens the inhibitory effect of calponin and markedly increases the effect of the 38 kD fragment of caldesmon. The results indicate similarity of molecular mechanisms of the regulation of muscle contraction by calponin and the actin-binding fragment of caldesmon. It is proposed that the regulation of smooth muscle contraction by calponin and caldesmon is carried out via the inhibition of the formation of the stage AM in ATP hydrolysis cycle.

[The effect of calponin on the rate of actin filament movement]. / Vliianie kal'ponina na skorost' dvizheniia aktinovykh nitei.

The effect of calponin on the velocity of actin filaments sliding over skeletal and phosphorylated smooth myosins was studied by in vitro mobility assay. It was found that calponin, being part of an actin filament, inhibits the average velocity of thin filaments movement. The analysis of histograms of the velocities showed that in the presence of calponin, actin filaments are capable to slow down the sliding, stop moving and move with high velocity, characteristic of calponin-free filaments. Tropomyosin weakens the inhibiting effect of calponin. It is supposed that calponin inhibits the sliding of thin filaments in more "all or none" fashion.

Inhibition of smooth muscle actomyosin ATPase by caldesmon is associated with caldesmon-induced conformational changes in tropomyosin bound to actin.

The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local conformational changes in different regions of the tropomyosin molecule was determined on the basis of changes in the excimer fluorescence (excited dimer of pyrene) formed in homodimers of tropomyosin isoforms. In the absence of actin, excimer fluorescence from the pyrene at Cys-190 of gamma-tropomyosin homodimer decreased in a simple manner on the addition of CaD, whereas the excimer from the Cys-36 of beta-tropomyosin homodimer exhibited a biphasic change, suggesting that additional weak binding sites exist near Cys-36. In the presence of actin, CaD-induced changes in the excimer fluorescence of pyrene-tropomyosin were observed only with Cys-36, and this change was associated with an inhibition of actin-activated myosin ATPase. A competition study with unlabeled tropomyosin isoforms indicated that the different excimer changes exhibited by beta- and gamma-tropomyosin in the presence of CaD were due to conformational changes in different regions of the tropomyosin molecule and not to differences in their affinities for CaD. Experiments with recombinant CaD mutants derived using the baculovirus expression system showed that the inhibition of tropomyosin potentiation of actomyosin ATPase by CaD requires the regions between residues 728-756 and 718-727 on the CaD molecule, although the latter region was sufficient for direct interaction with tropomyosin.

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity.

The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20 +/- 0.046 microns s-1 at 30 degrees C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.

Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system.

Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5'-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3'-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 micrograms per 10(6) cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36-48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca(2+)-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.

Myosin I from mammalian smooth muscle is regulated by caldesmon-calmodulin.

A single-headed monomeric myosin (myosin I) was isolated from pig urinary bladder smooth muscles and purified to homogeneity. Myosin I from smooth muscle is composed of a 110-kDa heavy chain and three 17-kDa light chains. The heavy chain from smooth muscle myosin I does not cross-react with the antibody against conventional myosin (myosin II) from smooth muscle, but it does show antigenic similarity to adrenal medulla myosin I heavy chain. The light chain from smooth muscle myosin I is similar to calmodulin in molecular weight, amino acid composition, and migration on SDS-polyacrylamide gel electrophoresis in the presence of Ca2+. The high salt ATPase activity of myosin I in the presence of CaCl2 is higher than that in K(+)-EDTA. Smooth muscle actin causes a 5-10-fold activation of the Mg-ATPase activity of myosin I. In the presence of Ca2+, exogenous calmodulin enhances the actin-activated ATPase activity of myosin I, and the increased activity is associated with the binding of exogenous calmodulin to myosin I heavy chain. A maximum of 4 mol of light chains/mol of myosin I heavy chain is observed in the presence of exogenous calmodulin. Caldesmon, a calmodulin/actin-binding protein, inhibits the actin-activated ATPase activity of myosin I. This inhibition is reversed by exogenous calmodulin in the presence of Ca2+. The actin activation of myosin I ATPase exhibits around 50% Ca2+ sensitivity in the presence of exogenous calmodulin. When caldesmon is bound to actin, Ca2+ sensitivity is increased to 80% in the presence of calmodulin. Therefore, smooth muscle caldesmon, which is thought to play a role in the regulation of actin activation of myosin II, also regulates the actin activation of myosin I ATPase in smooth muscle.

Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder.

Hypertrophy of the urinary bladder was produced in rabbit by partial ligation of the urethra. Electrophoresis of the bladder smooth muscle myosin on highly porous (3.5-7% gradient) SDS-polyacrylamide gel revealed two heavy chain isoforms, SM-1 and SM-2 with approximate molecular weights of 204,000 and 200,000, respectively. The ratio of the SM-2 to SM-1 heavy chain is 3:1 for myosin isolated from normal bladder smooth muscle, and this ratio changes to about 1:1 in hypertrophied bladder. Despite a change in the ratio of SM-2 to SM-1, the myosin ATPase and the actin-activated ATPase activities are not altered in response to hypertrophy.

The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.

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.

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin.

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.