Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.     

Subchloroplastic localization of NAD kinase activity: evidence for a Ca2+, calmodulin-dependent activity at the envelope and for a Ca2+, calmodulin-independent activity in the stroma of pea chloroplasts

Chloroplasts were prepared from pea seedlings and tested for NAD kinase activity. More than half of a Ca²⁺, calmodulin-dependent activity and most of a Ca²⁺, calmodulin-independent activity of the homogenate were associated with chloroplasts. The Ca²⁺, calmodulin-dependent activity could be detected by adding Ca²⁺ and calmodulin to the incubation medium containing intact chloroplasts. This activity could not be separated from the chloroplasts by successive washes or by phase partition in aqueous two-polymer phase systems. After chloroplasts fractionation, the Ca²⁺, calmodulin-dependent NAD kinase activity was localized at the envelope, and the Ca²⁺, calmodulin-independent activity was recovered from the stroma. In view of these results and of a previous report [Simon (1982) Plant Cell Rep. 1, 119–122] the occurrence and presumed role of calmodulin in the chloroplast are discussed.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.     

A Method for the Purification of Phospho(Tyr)calmodulin Free of Nonphosphorylated Calmodulin

Phosphocalmodulin has been shown to have a differential biological activity compared to nonphosphorylated calmodulin when assayed on a variety of calmodulin-dependent systems. However, the phosphocalmodulin preparations used so far in those experiments were not necessarily free of nonphosphorylated calmodulin. Therefore, the results obtained may not unquestionably show the real effect of pure phosphocalmodulin on the systems under study. To solve this problem, we describe here a method for the purification of phospho(Tyr)calmodulin free of nonphosphorylated calmodulin. The procedure consists of the following steps: (i) phosphorylation of calmodulin by a fraction enriched in epidermal growth factor receptor tyrosine kinase from rat liver isolated by calmodulin affinity chromatography, (ii) isolation of a calmodulin/phosphocalmodulin mixture by Ca(2+)-dependent chromatography in phenyl-Sepharose, (iii) purification of phospho(Tyr)calmodulin using an anti-phosphotyrosine antibody immobilized in agarose upon elution with phenyl phosphate, and (iv) removal of phenyl phosphate from the phospho(Tyr)calmodulin preparation by filtration chromatography in a Bio-Gel P-2 column. The obtained phospho(Tyr)calmodulin preparation was highly pure and essentially free of nonphosphorylated calmodulin because of the use of anti-phosphotyrosine affinity chromatography. We demonstrate that this ultrapure phospho(Tyr)calmodulin preparation is totally incapable of activating the calmodulin-dependent cyclic nucleotide phosphodiesterase. In contrast, when a nonpurified phospho(Tyr)calmodulin preparation was used a partial activation of this enzyme was observed.     

Calmodulin, activated cyclic nucleotide phosphodiesterase, microtubules, and vinca alkaloids

Calmodulin 1.8 and 10.6 microM, inhibited the polymerization of bovine brain microtubules by 30 and 50%, respectively. Two 55,000- to 68,000-dalton calmodulin-binding protein as well as calmodulin-dependent and -independent phosphodiesterases (PDE) were found associated with microtubule proteins. Among the antimicrotubule drugs, such as colchicine, podophyllotoxin, griseofulvin, and vinca alkaloids (vinblastine, desacetylvinblastine amide, and vincristine), the vinca alkaloids were selective inhibitors of calmodulin-activated PDE activity. This action of vinca alkaloids resides in the catharanthine moiety of vinblastine molecule. An alpha 2 inhibitor, yohimbine, affects the microtubules, and a series of alpha-adrenoceptor blocking agents were examined for their effects on calmodulin-dependent PDE. The relative order of the potency is phenoxybenzamine = dibenamine greater than phentolamine greater than yohimbine greater than prazosin greater than tolazoline, and the first four drugs in this series were selective inhibitors of calmodulin action. Inasmuch as phenoxybenzamine and dibenamine are alkylating agents, the effects of antineoplastic alkylating agents on the calmodulin action were also examined. Busulphan, melphalan, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and streptozotocin (up to 4 mM) were not selective inhibitors of calmodulin action. Maytansine, a vinca alkaloid-type antimicrotubule agents as well as an alkylating agent, selectively inhibited the calmodulin with a potency similar to vincristine. In addition, phenoxybenzamine affected Ca2+-dependent fluorescence induced by the interaction between calmodulin and hydrophobic fluorescent probes, whereas vinblastine was ineffective. However, the binding of vinblastine to calmodulin is calcium dependent. Studies such as these suggest the importance of physical and structural considerations in drugs binding to calmodulin as well as at least two different binding sites for drugs on calmodulin.     

A genetic approach to reveal the action of the opiate receptor in selected neuroblastoma-glioma cells. Interaction with alpha-adrenoceptors, calmodulin and Ca2+-ATPase

Three clones of neuroblastoma-glioma cells that contain low amounts of calmodulin were selected from the NG108-15 cells after several treatments with high concentrations of chlorpromazine. Purified membranes of the three clones had decreased numbers of both alpha-adrenergic and opiate receptors, monitored with [3H]yohimbine and [3H,D-Ala2]methionine encephalinamide, respectively. No changes were observed in the affinity of these radioactive ligands to the receptors of the selected cells as compared to the parent cells. Addition of bovine brain calmodulin did not affect the binding of [3H,D-Ala2]methionine encephalinamide to the membranes of the selected cells and they had the same number of acetylcholine receptors, determined with 1-quinuclidinyl-[phenyl-4-3H]-benzilate, as the parent NG108-15 cells. The basal ATPase activity in the membranes of the selected cells was 35-50% of the parent cells, with a decreased V value and no significant change in the affinity constant Ka to ATP. Addition of Ca2+ to the purified membranes increased the V of the ATPase in the selected as well as the parent cells but the V of the selected cells remained lower than that of the parent cells. Ca2+ had no effect on the Ka to ATP in either cell type. The Ca2+-dependent ATPase activity of both the parent and the selected cells was also calmodulin-dependent dependent since it was blocked in vitro by chlorpromazine. The co-regulation of opiate and adrenergic receptors and their interaction with calmodulin and Ca2+-ATPase is discussed in view of recent observations indicating biochemical and physiological association between opiates, Ca2+ and adrenergic compounds.     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.     

Phosphorylation of caldesmon

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulin-dependent protein kinase II.     

Calmodulin Involvement in Stress- and Corticosterone-Induced Down-Regulation of Cyclic AMP-Generating Systems in Brain

Manipulation of the hypothalamic-pituitary-adrenal axis selectively alters alpha-adrenergic potentiation of the cyclic AMP response to beta-adrenergic receptor stimulation in rat cerebral cortex. Calcium has been implicated in this alpha-receptor-mediated response, which may involve activation of phospholipases A2 and C and/or calmodulin-dependent adenylate cyclase. We therefore investigated the effects of stress and corticosterone (CORT) on membrane calmodulin-dependent adenylate cyclase and noradrenaline-stimulated cyclic AMP accumulation in brain slices. Repeated stress for 21 days selectively attenuated the adenylate cyclase response to calcium/calmodulin in cerebral cortex membranes, without affecting basal or forskolin-stimulated enzyme activity. There was no such effect in hippocampal membranes. The same pattern of response was elicited by daily CORT injection (50 mg/kg s.c.) for 21 days, while vehicle injection had no effect. CORT in the drinking water (400 micrograms/ml) elicited the same reduction of body weight as CORT injections, but had no effect on calmodulin adenylate cyclase. In parallel with calmodulin adenylate cyclase, cyclic AMP accumulation elicited by noradrenaline in slices of cerebral cortex was suppressed by both stress and daily CORT injections, with smaller effects observed with CORT in the drinking water. Unlike calmodulin adenylate cyclase, noradrenaline-stimulated cyclic AMP accumulation in hippocampus showed the same suppression as that in cerebral cortex. These results are discussed in relation to the differential mode of coupling of alpha-adrenergic receptors to cyclic AMP-generating systems between brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular coupling via limiting calmodulin

Measurements of cellular Ca2+-calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric-oxide synthase and the catalytic activity of the enzyme both are increased approximately 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca2+ and Ca2+-calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (Kd = 2 nm) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca2+-calmodulin concentrations produced and an increase in the [Ca2+]50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca2+ pump activity. An identical reduction in pump activity is produced by expression of a high affinity (Kd = 0.3 nm) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Interaction of Cd2+ with the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of human erythrocyte ghosts

Treatment of erythrocyte ghosts with micromolar concentrations of Cd2+ results in a noncompetitive inhibition of the calmodulin-dependent (Ca2+ + Mg2+)-ATPase activity. Higher concentrations of Cd2+ are required for inhibition of the (Ca2+ + Mg2+)-ATPase activity of calmodulin-depleted ghosts. The interaction of Cd2+ is time-dependent with an apparent rate constant around 0.12/min. The inhibition is relieved by addition of EGTA with a rate constant around 0.15/min. If Cd2+ is allowed to interact with calmodulin prior to the association of the protein with the ghosts, the inhibition is mainly competitive. The results suggest that the inhibitory effect caused by Cd2+ is due to an interaction with calmodulin. The slow interaction of Cd2+ suggests that calmodulin bound to the (Ca2+ + Mg2+)-ATPase is inaccessible to Cd2+.     

Changes in conformation of spin-labeled calmodulin by phospholipids

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.     

Inhibition of Calmodulin-Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.     

Insulin-receptor phosphotyrosyl-protein phosphatases.

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.     

The use of parameters of non-uniform widening of probe fluorescence spectra in the study of conformation changes in Ca2+-binding proteins

The parameters of inhomogeneous broadening in the fluorescence spectra of 1-anilinonaphthalene-8-sulfonate and N-phenyl-1-naphthylamine, recorded in the systems with respective proteins, have been analyzed in order to shed light on the mechanism of interaction between Ca2+ ions and calmodulin, troponin C and parvalbumin. It was shown that only calmodulin and troponin C but not parvalbumin bind calcium ions with concomitant formation of hydrophobic sites that are responsible for interaction with the "executor enzymes". The relative pools of the probes adsorbed in the hydrophobic sites and polarity of the latter were assessed. These parameters in calmodulin obtained from the brain of spontaneously hypertensive rats or normotensive rats do not differ. It was established that trifluoroperazine and verapamil inhibit the calmodulin-dependent enzymes by essentially different mechanisms. Trifluoroperazine diminishes the relative pool of the adsorbed probe and enhances the polarity of the calmodulin binding sites, whereas verapamil affects these parameters in the opposite direction.     

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.     

Identification of beta-endorphin residues 14-25 as a region involved in the inhibition of calmodulin-stimulated phosphodiesterase activity

The inhibition of the calmodulin-mediated stimulation of bovine brain cyclic nucleotide phosphodiesterase activity (3':5'-cyclic adenosine monophosphate 5'-nucleotidohydrolase, EC 3.1.4.17) by the 31-residue opiate peptide beta-endorphin has been investigated. Using conditions in which porcine brain calmodulin (6 nM) is limiting (i.e., to give a 3-fold, Ca2+-dependent stimulation of enzymic activity toward cyclic guanosine monophosphate), the domain of beta-endorphin responsible for the inhibition was mapped by using a series of deletion peptides. beta-Endorphin exhibited an ED50 of several micromolar under the conditions employed, and several amino-terminal deletion peptides were essentially as inhibitory as the parent peptide. Methionine enkephalin and various carboxy-terminal deletion peptides had no demonstrable effect at concentrations of 100-200 microM. Peptides 1-25 and 1-27 (C' fragment) inhibited the calmodulin-dependent activity of phosphodiesterase, but higher concentrations were required than of beta-endorphin. Studies using combined amino- and carboxy-terminal deletion peptides demonstrate that peptide 14-25 was the shortest peptide examined that was capable of inhibiting calmodulin stimulation of phosphodiesterase activity under the conditions used. There was no evidence to indicate that the amino-terminal region comprising residues 1-13 of beta-endorphin contributes to the measured inhibition of calmodulin-stimulated enzymic activity. The circular dichroic spectra of calmodulin, beta-endorphin, and mixtures of the two were obtained, and the ellipticity of the peptide-protein mixtures at 221 nm exceeded that expected by assuming simple additivity. This finding is consistent with a direct interaction of beta-endorphin with calmodulin which seems to lead to enhanced helicity of one or both components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a Role of Calmodulin in Calcium-Induced Noradrenaline Release from Permeated Synaptosomes: Effects of Calmodulin Antibodies and Antagonists

Abstract: The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca²⁺-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca²⁺/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca²⁺-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca²⁺-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca²⁺-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca²⁺-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca²⁺/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.