Interaction of calmodulin with troponin I and the troponin-tropomyosin-actin complex. Effect of Ca2+ and Sr2+ ions.

In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.     

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.     

Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.     

Thermodynamic study of domain organization in troponin C and calmodulin

Intramolecular melting of troponin C, calmodulin and their proteolytic fragments has been studied microcalorimetrically at various concentrations of monovalent and divalent ions. It is shown by thermodynamic analysis of the experimentally determined excess heat capacity function that the four calcium-binding domains in these two related proteins are not integrated into a single co-operative system, as would be the case if they formed a common hydrophobic core in the molecule, but still interact with each other in a very specific way. There is a positive interaction between domains I and II, which is so strong that they actually form a single co-operative block. The interaction between domains III and IV is positive also, although much less pronounced, while the interaction between the pairs of domains (I and II) and (III and IV) is negative, as if they repel each other. The structure of the co-operative block of domains I and II at room temperature does not depend noticeably on the ionic conditions, which influence its stability to a small extent only. The same applies to domain IV of calmodulin, but in troponin C this domain is unstable in the absence of divalent ions, in solutions of low ionic strength. In both proteins, the least stable is domain III, which forms a compact ordered structure at room temperature only in the presence of Ca2+. In troponin C, calcium ions can be replaced by magnesium ions, although the compact structure of domain III formed by these two ions does not seem to be quite identical. Thus, at conditions close to physiological, with regard to temperature and ionic strength, the removal of free Ca2+ from the solution induces in both proteins a reversible transition of domain III to the non-compact disordered state. This dramatic Ca2+-induced change in the domain III conformation in troponin C and calmodulin might play a key role in the functioning of these proteins as a Ca2+-controlled switch in the molecular mechanisms of living systems.     

Role of calmodulin in the activation of tryptophan hydroxylase

Tryptophan hydroxylase can be activated 2.0- to 2.5-fold in vitro by ATPa dn Mg2+. This apparent phosphorylation effect is not dependent on cyclic nucleotides but is dependent on the presence of calcium. The activation of tryptophan hydroxylase by ATP-Mg2+ reduces the apparent Km of the enzyme for its cofactor, 6-methyltetrahydropterin, from 0.21 to 0.09 mM. The addition of certain antipsychotic drugs known to bind to calmodulin in a phosphorylation reaction mixture prevents the activation to tryptophan hydroxylase by ATP-Mg2+ in the concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Preparation of calmodulin-free tryptophan hydroxylase by affinity chromatography on fluphenazine-Sepharose 4B yields an enzyme that is no longer activated by ATP-Mg2+, whereas the readdition of calmodulin to a calmodulin-free enzyme restores the responsiveness of tryptophan hydroxylase to ATP-Mg2+. This restoration is dependent on Ca2+. Taken together, these results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobe

We have studied the calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobes: one, squid calmodulin (SCaM), similar to bovine brain calmodulin (BCaM), the other, squid calcium-binding protein (SCaBP), distinct (Head, J.F., Spielberg, S., and Kaminer, B. (1983) Biochem J. 209, 797-802). Equilibrium dialysis measurements on the squid proteins (and BCaM) were made at 100 mM KCl in the presence and absence of 3 mM Mg2+, and at 400 mM KCl in the presence of 3 mM Mg2+, which more closely resembles the conditions in the squid. SCaM, SCaBP, and BCaM each bind a maximum of 4 Ca2+ ions/molecule of protein under the ionic conditions tested. SCaBP has a higher affinity than SCaM or BCaM for Ca2+ at 100 mM KCl in the absence of Mg2+. However, in the presence of Mg2+, half-maximal binding to SCaBP occurs at a similar pCa value to that observed with calmodulin. Increasing the KCl concentration reduces the affinity of all three proteins for Ca2+. UV absorption measurements showed that the binding of 4 Ca2+ ions/molecule is necessary to complete spectral changes in SCaBP, compared to two for the calmodulins. While Ca2+ causes perturbations in aromatic chromophores in SCaM and SCaBP, Mg2+ causes a significant perturbation only in SCaBP. These Mg2+-induced changes differ qualitatively from those induced by Ca2+.     

Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Squid visual arrestin: cDNA cloning and calcium-dependent phosphorylation by rhodopsin kinase (SQRK)

Arrestin binding to rhodopsin is one of the major mechanisms of termination of photoresponses in both vertebrates and invertebrates. Here we report the cDNA cloning and characterization of a 48-kDa visual arrestin from squid (Loligo pealei). The cDNA encoded a protein that had 56-64% amino acid sequence similarity to reported arrestin sequences. This protein does not encode any distinct modular domains but contains five fingerprint regions that have been identified within arrestins. Antibodies raised to the recombinant arrestin protein detected arrestin expression only in the eye and recognized a doublet in photoreceptor membranes, representing unphosphorylated and phosphorylated arrestin. In squid eye membranes, arrestin was phosphorylated in a Ca2+-dependent manner and this phosphorylation was inhibited by antibodies raised against squid rhodopsin kinase, but not by inhibitors of protein kinase C or calmodulin kinase. Addition of purified squid rhodopsin kinase to washed rhabdomeric membranes resulted in phosphorylation of rhodopsin, and arrestin was also phosphorylated when calcium was present. This is the first report of a rhodopsin kinase phosphorylating an arrestin substrate, and suggests a dual role for this kinase in the inactivation of the squid visual system.     

Effect of neurotropic drugs on calmodulin and troponin C-dependent processes

The effects of neurotropic compounds on Ca-binding proteins (calmodulin, troponin C) were investigated. It was shown that the majority of neuroleptics of the phenothiazine group effectively interact with the both proteins and inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+-activated actomyosin. ATPase. Neuroleptics of the butyrophenone group as well as imipramine and diphenehydramine having a low efficiency interact only with calmodulin. Methophenazine, a phenothiazine neuroleptic, being an effective inhibitor of calmodulin and of calmodulin-dependent phosphodiesterase, does not influence troponin C or Ca-dependent actomyosin ATPase. Therefore, this compound may be used as a convenient tool in the study of processes controlled by these Ca-binding proteins. It is concluded that troponin C possesses Ca-dependent sites which bind pharmacological agents structurally similar to that of calmodulin. However, these sites bind pharmacological agents with a low efficiency and exhibit selectivity towards certain drugs. Despite the obvious homology of the both Ca-binding proteins, i.e., calmodulin, troponin C, their effects on the processes under their control appear to be selective.     

Clathrin light chains are calcium-binding proteins

Clathrin light chains have been purified to near homogeneity. When analyzed by sodium dodecyl sulfate gel electrophoresis followed by silver stain for proteins, no bands corresponding to light chains were detected. As calmodulin and troponin C are known to behave in the same manner on silver staining, the possibility that clathrin light chains were Ca2+-binding proteins was investigated. Light chains fixed to nitrocellulose filters were found to bind 45Ca2+ in the presence of 5 mM Mg2+. The Ca2+-binding capacity of the light chains was further investigated, using gel filtration and equilibrium dialysis. The light chains were shown to bind, in the presence of 3 mM Mg2+, 1 mol of Ca2+ per mol of light chain with a Kd of 25-55 microM. Nitrocellulose binding and gel filtration studies showed that light chains present in triskelions are still capable of binding Ca2+, in this case with a calculated Kd of 45 microM.     

Effect of pH on Ca-binding properties of calmodulin and on its interaction with Ca-dependent phosphodiesterase of cyclic nucleotides

The fluorescence of dansyl immobilized on bovine brain calmodulin is sensitive to Ca2+. This effect is due to Ca2+ attachment to specific Ca2+-binding sites of calmodulin and is maintained within a wide range of pH. The native and dansyl-modified calmodulin preparations exert similar activating effects on Ca-dependent phosphodiesterase of cyclic nucleotides and have practically the same affinity for the enzyme. Using fluorescence measurements of the calmodulin--dansyl conjugate, it was shown that the decrease of pH from 9.0 down to 6.0 gradually decreases the constant of Ca2+ binding to calmodulin from 1.5 . 10(10) M-1 to 1.6 . 10(6) M-1. This decrease of pH does not affect the calmodulin affinity for phosphodiesterase. The activating effect of calmodulin on phosphodiesterase is more pronounced at acidic pH values (6.0-7.0) than at alkaline pH values (8.0-9.0).     

A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding

The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.     

Calmodulin and troponin C: affinity chromatographic study of divalent cation requirements for troponin I inhibitory peptide (residues 104-115), mastoparan and fluphenazine binding

The different conformations induced by the binding of Mg2+ or Ca2+ to troponin C (TnC) and calmodulin (CaM) results in the exposure of various interfaces with potential to bind target compounds. The interaction of TnC or CaM with three affinity columns with ligands of either the synthetic peptide of troponin I (TnI) inhibitory region (residues 104-115), mastoparan (a wasp venom peptide), or fluphenazine (a phenothiazine drug) were investigated in the presence of Mg2+ or Ca2+. TnC and CaM in the presence of either Ca2+ or Mg2+ bound to the TnI peptide 104-115. The cation specificity for this interaction firmly establishes that the TnI inhibitory region binds to the high affinity sites of TnC (most likely the N-terminal helix of site III) and presumably the homologous region of CaM. Mastoparan interacted strongly with both proteins in the presence of Ca2+ but, in the presence of Mg2+, did not bind to TnC and only bound weakly to CaM. Fluphenazine bound to TnC and CaM only in the presence of Ca2+. When the ligands interacted with either proteins there was an increase in cation affinity, such that TnC and CaM were eluted from the TnI peptide or mastoparan affinity column with 0.1 M EDTA compared with the 0.01 M EDTA required to elute the proteins from the fluphenazine column. The interaction of these ligands with their receptor sites on TnC and CaM require a specific and spatially correct alignment of hydrophobic and negatively charged residues on these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.     

Hydrophobic regions function in calmodulin-enzyme(s) interactions

Certain naturally occurring lipids (phosphatidylinositol, phosphatidylserine, arachidonic acid) and sodium dodecyl sulfate activate at least two calmodulin-dependent enzymes, bovine brain 3':5'-cyclic nucleotide phosphodiesterase and chicken gizzard myosin light chain kinase in the absence of Ca2+. 2-p-Toluidinyl-naphthalene-6-sulfonate (TNS), which is often used as a probe for hydrophobic groups of proteins, inhibits these two calmodulin-dependent enzymes. Kinetic analysis of inhibition of chicken gizzard myosin kinase by TNS revealed a competitive fashion against calmodulin-induced activation. The interaction between TNS and purified bovine brain calmodulin as demonstrated in the appearance of TNS fluorescence in the presence of 3 microM or more of calcium ion was not observed in the presence of 2 mM EGTA. This suggests that TNS is able to bind to calmodulin in the presence of Ca2+. Moreover, a calmodulin-interacting agent N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide suppressed the TNS fluorescence induced by complex formation with calmodulin in the presence of Ca2+. These results suggest that when Ca2+ binds to the high affinity sites of calmodulin, it induces a conformational change which exposes hydrophobic groups, and the calmodulin is then capable of activating calmodulin-dependent enzymes. We propose that hydrophobic properties of Ca2+-calmodulin are important for the activation of Ca2+-calmodulin-dependent enzymes.     

Ion binding to calmodulin

Summary Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca²⁺ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca²⁺ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca^2– binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.Abbreviations CaBP calcium-binding protein - ICaBP the vitamin D-dependent intestinal Cat+binding protein - S-100 the glial S-100 protein - RLC the phosphorylatable myosin regulatory light chain - CaM calmodulin - Pa parvalbumin - TnC troponin C - TnI troponin I - Hepes N-2-hydroxyethylpipezarine, N-2-ethane-sulfonic acid - W7 N-(6-Aminohexyl)-5-chloro-l-Naphtalene sulfonamide - SDS sodium dodecyl sulfate - NMR nuclear magnetic resonance