Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.     

A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.     

Organic acids prevent aluminum-induced conformational changes in calmodulin

At a molar excess of 10:1 for [citrate]/[calmodulin], citrate can prevent aluminum binding to calmodulin when present in the protein solution in micromolar concentration, as determined by fluorescence and circular dichroism spectroscopy. In contrast, citrate is only partially effective in restoring calmodulin to its native structure once the aluminum-calmodulin complex (3:1) is formed, as measured by the α-helix content of the protein. Considering the magnitude of the stability constant of the citrate-aluminum chelate, citrate and perhaps other carboxylic acids may protect calmodulin, and thus cells, from toxic aluminum ions.     

Calmodulin dependent formation of membrane potential in barley root plasma membrane vesicles: A biochemical model of aluminum toxicity in plants

Micromolar concentrations of aluminum ions interfere with calmodulin-stimulated, membrane bound ATPase activity which plays a role in the maintenance of the transmembrane potential of plasma membrane enriched vesicles isolated from barley roots. Calmodulin appears to be the major target for aluminum interaction resulting in pronounced changes in the exposure of a large, hydrophobic surface on this protein as determined with a fluorescent, hydrophobic surface probe. At a molar ratio of 3:1 [aluminum]/[calmodulin], the calmodulin stimulated enzymatic activity, probably associated with a Ca²⁺+ Mg²⁺ATPase, is about 95% inhibited. Aluminum induced changes in calmodulin structure are reflected in reduced formation of the membrane potential when assayed with a fluorescent potential probe, oxonol VI. We hypothesize that the aluminum caimodulin complex represents a primary lesion in toxic responses of plants to this metal.     

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.     

Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.     

Temperature and thiol-induced desensitization of a Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung

Ca2+-sensitivity of sheep lung cyclic-3',5'-nucleotide phosphodiesterase is provided by endogenous tightly bound calmodulin. The calcium sensitivity of a highly purified enzyme was desensitized by increasing the assay temperature. It could also be desensitized to Ca2+-activation by thiols such as dithiothreitol. The thiol-induced desensitization could be partially reversed by dialysis and almost completely reversed by dilution. The results presented in this paper indicate that thiols are possibly involved in the interaction of calmodulin with cyclic-3',5'-nucleotide phosphodiesterase. This is the first report on temperature and thiol-induced desensitization of Ca2+-sensitivity of a cyclic-3',5'-nucleotide phosphodiesterase.     

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.     

Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine--Sepharose affinity chromatography.

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.     

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae

Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.     

Interaction of calcium ions with α-elastin: An equilibrium dialysis,circular dichroism,and microcalorimetric study

The interaction of calcium ions with α-elastin has been investigated by equilibrium dialysis, CD, and microcalorimetric techniques. Consistent with literature data, it was found that the interaction in water is very poor. In trifluoroethanol (TFE), equilibrium dialysis experiments showed that calcium ions bind to ～-elastin with an association constant of ～250 L × mol^?1. Such a figure is not consistent with highly specific, highly selective binding. It was also found that the CD response is not directly proportional to the amount of bound calcium but depends on the protein concentration. From microcalorimetric experiments it was found that the heat effect relative to the binding process is of the order of 1.9 kcal/g ion. From this figure and from the binding constant, a positive ΔS value of about 17 e.u. was evaluated, leading to the conclusion that the binding process is entropy driven. From microcalorimetric measurements a ΔH of 1.5 kcal/residue was found for the calcium-induced conformational transition of the protein.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.     

Mastoparan binding induces Ca(2+)-transfer between two globular domains of calmodulin: a 1H NMR study.

The interaction between calmodulin and mastoparan at various concentrations of calcium ions was studied by 1H NMR. It was found that at lower mastoparan concentrations 1 mol of mastoparan binds to both the C-terminal-half and N-terminal-half regions of calcium-saturated calmodulin. The mastoparan affinity is much greater for the C-terminal-half region than for the N-terminal-half region. At higher mastoparan concentrations, a further 1 mol of mastoparan binds to the N-terminal-region of calcium saturated calmodulin. The results can be interpreted in terms of the assumption that the N-terminal-half region of calmodulin with mastoparan has a higher calcium ion affinity than the C-terminal-half region without mastoparan. It is suggested that calcium ions transfer from the C-terminal-half region of calmodulin without mastoparan to the N-terminal-half region of calmodulin with mastoparan. This calcium ion transfer is discussed from the viewpoint of enzyme activation by calmodulin.     

Calcium effects on calmodulin lysine reactivities

The differential reactivities of individual lysines on porcine testicular calmodulin were determined by trace labeling with high specific activity [3H]acetic anhydride as a function of the molar ratio of Ca2+ to calmodulin. In progressing from the Ca2+-depleted form of the protein to a Ca2+:calmodulin molar ratio of 5:1, six of the seven lysyl residues exhibited a modest 1.5- to 3.0-fold increase in reactivity. Lys 75, in contrast, was enhanced in reactivity greater than 20-fold. When the change in reactivity of each lysine was normalized as a percentage of the maximum change, most of the residues were found to fall into two distinct classes. One class, comprising lysines 94 and 148 from the two carboxy terminal Ca2+-binding domains 3 and 4, respectively, exhibited about 90% of their reactivity change when the Ca2+:calmodulin molar ratio was 2:1, and these residues were perturbed very little upon further addition of Ca2+. The other class, encompassing lysines 13, 21, and 30 from the amino terminal domain 1 and Lys 75 from the extended helix connecting the two globular lobes of calmodulin, underwent most of their overall reactivity change (55-70%) between 2 and 5 equivalents of Ca2+ per mol of calmodulin. Lys 77 was distinct in its pattern of change, undergoing approximately equal changes with each Ca2+ increment. These results are consistent with a model where Ca2+ first binds to the two carboxy terminal sites of calmodulin with no apparent preference, concomitant with minor alterations in the microenvironments of lysines in the unoccupied amino terminal domains. The third and fourth Ca2+ ions then bind to these latter two domains, again with no evidence of preference, with little change in the lysine reactivities at the carboxy terminus of the molecule. The environments of groups in the central helix appear to undergo changes in a manner that reflects their proximity to the amino and carboxy terminal domains. In the course of this work, it was found that Lys 94 in apocalmodulin is specifically perturbed by the addition of EGTA, suggesting that the chelating agent may interact with calmodulin at or near the third Ca2+-binding domain.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Subcellular distribution of potassium, sodium, magnesium, calcium and chloride in cerebral cortex

The subcellular distributions of potassium, sodium, magnesium, calcium and chloride have been determined for rabbit cerebral cortex. After homogenization and differential centrifugation, the following percentages of ions were associated with the particulate fraction (nuclear, mitochondrial, synaptic vesicles, and microsomal): (a) 19% of the total potassium; (b) 22% of the total sodium; (c) 77% of the total calcium; (d) 69% of the total magnesium; and (e) less than 2% of the total chloride. However, the sum of the potassium and sodium content in each of the particulate fractions was greater than the sum of the calcium and magnesium content. After hypo-osmotic shock of the crude mitochondrial fraction (M_T), more sodium than potassium (μmol/g wet wt.) was associated with the mitochondrial (M₁) and synaptic vesicle (M₂) fractions. The molar ratio of sodium to potassium was 1·4 for M₁ and 4·5 for M₂. The association of ²²Na⁺ with the particulate fractions was further studied by the method of equilibrium dialysis. The data from both types of experiments indicate that a large fraction of the sodium in cortical tissue appears to be in a bound state.     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

Brush-border calmodulin. A major component of the isolated microvillus core

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons.The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.