Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.     

Two trifluoperazine-binding sites on calmodulin predicted from comparative molecular modeling with troponin-C

Among the known regulatory proteins that are conformationally sensitive to the binding of calcium ions, calmodulin and troponin-C have the greatest primary sequence homology. This observation has led to the conclusion that the most accurate predicted molecular model of calmodulin would be based on the X-ray crystallographic coordinates of the highly refined structure of turkey skeletal troponin-C. This paper describes the structure of calmodulin built from such a premise. The resulting molecular model was subjected to conjugate gradient energy minimization to remove unacceptable intramolecular non-bonded contacts. In the analysis of the resulting structure, many features of calmodulin, including the detailed conformation of the Ca2+-binding loops, the amino- and carboxy-terminal hydrophobic patches of the Ca2+-bound form, and the several clusters of acidic residues can be reconciled with much of the previously published solution data. Calmodulin is missing the N-terminal helix characteristic of troponin-C. The deletion of three residues from the central helical linker (denoted D/E in troponin-C) shortens the molecule and changes the orientation of the two domains of calmodulin by 60 degrees relative to those in troponin-C. The molecular model has been used to derive two possible binding sites for the antipsychotic drug trifluoperazine, a potent competitive inhibitor of calmodulin activity.     

A model of troponin-I in complex with troponin-C using hybrid experimental data: the inhibitory region is a beta-hairpin

We present a model for the skeletal muscle troponin-C (TnC)/troponin-I (TnI) interaction, a critical molecular switch that is responsible for calcium-dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin-C in complex with troponin-I, or in the ternary troponin-complex, have not yet delivered a high-resolution structure. Many groups have pursued different experimental strategies, such as X-ray crystallography, NMR, small-angle scattering, chemical cross-linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left-handed sense (Model L), and another that winds about TnC in a right-handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross-linking and FRET data. The inhibitory region TnI(95-114) is modeled as a flexible beta-hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a beta-hairpin region of the actin-binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the beta-hairpin is an important structural motif that communicates the Ca2+-activated troponin regulatory signal to actin.     

Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.     

Trifluoperazine binding to mutant calmodulins

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.     

Biochemical characteristics of the Mr 52,000 component of Akazara scallop troponin

Some biochemical properties of the Mr 52,000 component of Akazara scallop striated adductor troponin, which had been tentatively identified as troponin-I, were compared with those of rabbit troponin-I. Both the Mr 52,000 component and rabbit troponin-I together with rabbit tropomyosin inhibited the Mg-ATPase activity of rabbit reconstituted actomyosin to 1/10 of the original activity. The inhibition was neutralized by the addition of Akazara scallop and rabbit troponin-C or Patinopecten scallop calmodulin. The Mr 52,000 component and rabbit troponin-I were insoluble below 0.15 M KCl, but were solubilized by complexing with an equimolar amount of troponin-C or calmodulin. On alkaline urea-polyacrylamide gel electrophoresis, the Mr 52,000 component as well as rabbit troponin-I was found to form a stable complex with troponin-C or calmodulin in the presence of Ca2+.     

Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated threeDrosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating thein vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.This work was supported by grants from the NIH and Muscular Dystrophy Association to E. F.Sequences described herein have been filed in the EMBL and GenBank databases under Accession Numbers X76042, X76043, X76044, and X76045.     

Defining the region of troponin-I that binds to troponin-C

The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.     

Model for the interaction of amphiphilic helices with troponin C and calmodulin

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).     

Effect of trifluoperazine on cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro

We recently demonstrated that the preventive effect of trifluoperazine (a potent inhibitor of calmodulin, protein kinase C, and phospholipase A2) on cholesterol-induced atherogenic activity of smooth muscle cells was mediated through its ability to inhibit smooth muscle cellular DNA synthesis coupled with stimulation of LDL receptor synthesis. The present study addressed the effect of trifluoperazine on cholesterol metabolism of aortic SMCs enriched with cholesterol through the nonreceptor pathway and revealed that (a) TFP caused inhibition of cholesterol synthesis compared with control cells bathed with hypercholesterolemic medium alone. (b) The drug also caused inhibition of free cholesterol and cholesteryl ester accumulation within smooth muscle cells compared to control cells. These results demonstrate that the preventive effect of TFP on atherogenic activity of smooth muscle cells may also be due to its ability to affect the altered/modified cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.     

Structural and functional studies on Troponin I and Troponin C interactions

Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.     

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins.

Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.     

Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.

It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+ dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+ in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC.TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.     

Inhibition of myogenesis by trifluoperazine and compound 48/80

When trifluoperazine (TFP), a calmodulin antagonist, was given to chick or rat myoblasts in cultures, formation of multinucleated myotubes was inhibited. The inhibition of cell fusion by TFP in rat cultures prevents the normal increase in the amount of acetylcholine receptors (AChR) and creatine kinase (CK), while the levels of these proteins in chick muscle cultures are hardly affected. Another calmodulin antagonist, compound 48/80, inhibits fusion at doses that correspond closely to its antagonistic effects on calmodulin. Thus, our results suggest a possible role for calmodulin in the regulation of myoblast fusion, but not on the appearance of muscle proteins.     

Involvement of phosphoinositides,calmodulin and glyoxalase-I in cell proliferation in callus cultures of Amaranthus paniculatus

The effect of lithium and trifluoperazine (TFP) was studied on cell proliferation in callus cultures of Amaranthus paniculatus. TFP (20 μM) and lithium (40 mM) inhibited the callus growth by 50% and 80%, respectively. The inhibition by lithium was reversed by the addition of myoinositol (2.5 mM). Equimolar concentration of NaCl, as that of LiCl, had no significant effect on callus growth. The activity of calmodulin was inhibited by TFP as tested both by in vivo and in vitro experiments. The level of phosphatidylinositol (PI) in calli grown on lithium was lower than the calli grown on the medium containing inositol alone. The activity of the enzyme glyoxalase-I was inhibited by lithium and TFP. The inhibition of the enzyme activity by lithium was reversed by the addition of inositol. Possible involvement of phosphoinositide cycle, calcium and calmodulin in cell proliferation in in vitro cultures is suggested.     

Functional significance of the central helix in calmodulin

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.     

Effects of trifluoperazine, a calmodulin antagonist, on rabbit T- and B-cell responses to mitogens and antigen

Trifluoperazine (TFP), an inhibitor of the calcium-binding protein, calmodulin (CaM), was used to assess the role of calmodulin in the responses of rabbit lymphoid cells to stimulation with mitogen and antigen. After binding goat anti-rabbit Fab antibody, rabbit B cells lose their surface immunoglobulin (Ig) through endocytosis and then reexpress this protein during the next 24 hr. This reexpression was markedly inhibited by TFP. The brief and early addition of TFP markedly inhibited the increased [3H]thymidine (Tdr) uptake by rabbit T cells treated with concanavalin A and B cells exposed to anti-Fab. TFP greatly inhibited the induction by keyhole limpet hemocyanin (KLH) of the in vitro syntheses of antibody, Ig, and protein by KLH-primed lymph node cells (LNC). The earlier the TFP the greater was the inhibition of induction of these syntheses. However, once induced, synthesis and secretion of antibody were not inhibited by TFP. In striking contrast to the inhibition by TFP of the mitogenic and antigenic responses of lymphoid cells was the lack of effect of this drug on resting lymphocytes. Since TFP was not cytotoxic for either resting or mitogen- or antigen-stimulated LNC, it is highly unlikely that the observed inhibitory effects of this drug were due to its cytotoxicity. We postulate that an early signal for the activation of LNC proliferation, differentiation, and the syntheses of antibody, Ig, and protein involves a calcium-CaM-mediated reaction. Based on this work and that of others, the calcium-CaM complex may mediate an interaction between the ligand-occupied surface receptor and the cytoskeleton.     

Mutation of Lys-75 affects calmodulin conformation.

Some properties of synthetic calmodulin and its five mutants with replacement of Lys-75 were analyzed by means of electrophoresis, limited proteolysis and MALDI mass-spectrometry. A double mutant of calmodulin containing insert KGK between residues 80 and 81 and replacement of Lys-75 by Pro has a highly flexible central helix which is susceptible to trypsinolysis in the presence of Ca2+. Two mutants, K75P and K75E, having a distorted central helix demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg-90 and Arg-106 being the primary site of trypsinolysis of synthetic calmodulin are partially-protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between residues located in positions 71, 72 and 75. In the presence of Ca2+, the central helix of K75V is resistant to trypsinolysis. Mutations K75A and K75V decrease the rate of trypsinolysis of the central helix with a simultaneous increase of the rate of trypsinolysis in the C-terminal domain of calmodulin. It is concluded that the point mutation in the central helix has a long distance effect on the structure of calmodulin.     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.