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PubMed1989

The location of calmodulin in the pea plasma membrane.

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.

Animals↗
PubMed1989

Regulation of calmodulin-sensitive adenylate cyclase by the stimulatory G-protein, Gs.

Studies in bovine and rat brain membranes have suggested that calmodulin can potentiate neurotransmitter- and GTP-stimulated adenylate cyclase activities. To examine whether calmodulin and the stimulatory G-protein, Gs, are potentiative at a calmodulin-sensitive adenylate cyclase, Gs was purified from rabbit liver and reconstituted with a partially purified calmodulin-sensitive adenylate cyclase from bovine brain. Activated Gs (G*s) stimulated basal adenylate cyclase activity and enhanced the stimulation by calmodulin. The potentiation of the calmodulin-stimulated adenylate cyclase activity was dose-dependent with respect to G*s concentration. At the highest concentration of G*s tested (3 nM), a 2-fold enhancement of the calmodulin-stimulated adenylate cyclase activity was observed at all concentrations of calmodulin. The synergistic activation of adenylate cyclase by calmodulin and Gs was dependent on the presence of Ca2+ and occurred at physiologically relevant Ca2+ concentrations. The potentiation was not observed when either a nonactivated Gs or a mixture of activated Gi/Go was used. G*s was not able to stimulate or potentiate a calmodulin-stimulated adenylate cyclase purified from membranes pretreated with the nonhydrolyzable GTP analog, guanyl-5'-yl beta,gamma-imidodiphosphate. Photochemical cross-linking of 125I-calmodulin-diazopyruvamide to proteins having an Mr corresponding to the known Mr of adenylate cyclase was not enhanced by G*s. The results demonstrate that the guanyl nucleotide-dependent enhancement of calmodulin-stimulated adenylate cyclase activity is mediated by G*s and suggest that G*s modulates the enzymatic turnover of the calmodulin-stimulated activity.

Adenylyl Cyclases↗
PubMed1982

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Calmodulin is tightly associated with synaptic vesicles independent of calcium.

A protein in highly purified synaptic vesicles from elasmobranch electric organ is recognized by two specific antisera that recognize different determinants of calmodulin. The protein is indistinguishable from authentic calmodulin by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of calcium. It is tightly associated with the intact synaptic vesicle membrane even in the absence of calcium. It is on vesicles rather than membrane contaminants and cytoplasmically oriented since a calmodulin antibody (sheep anti-calmodulin antibody) immunoprecipitates at least 86% of intact synaptic vesicles. Surprisingly, another calmodulin antiserum (rabbit anti-calmodulin serum) specifically precipitates less than 20% of the intact vesicles. This antiserum (rabbit anti-calmodulin serum) also detects 4-15 times less calmodulin immunoreactivity than sheep anti-calmodulin antibody by radioimmunoassay of vesicles solubilized with nondenaturing detergents. The difference essentially disappears if the vesicle calmodulin is solubilized in sodium dodecyl sulfate. We suggest that the antigenic determinant recognized by rabbit anti-calmodulin serum is concealed in vesicle-associated calmodulin and may be involved in binding calmodulin to the vesicle.

Animals↗
PubMed1981

Characterization of a novel calmodulin from Dictyostelium discoideum.

We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Calmodulin stimulation and calcium regulation of smooth muscle adenylate cyclase activity.

The Ca2+-dependent regulation of the adenylate cyclase activity associated with microsomes isolated from bovine aortic smooth muscle has been studied. Calmodulin content of microsomal membranes employed in these studies was 80 +/- 14 ng/mg as determined by specific radioimmunoassay. In the absence of exogenous calmodulin, Ca2+ concentrations greater than 0.8 microM inhibited adenylate cyclase activity with one-half-maximal inhibition occurring at 2.5 microM Ca2+. In the presence of 5 or 9 microM bovine testis calmodulin, Ca2+ stimulated smooth muscle adenylate cyclase activity with one-half-maximal stimulation occurring at 0.2 microM for both 5 and 9 microM calmodulin. Calmodulin stimulation was observed between 0.1 and 0.8 microM Ca2+. Despite the presence of calmodulin, Ca2+ concentrations greater than 0.8 microM were inhibitory to smooth muscle adenylate cyclase activity. However, calmodulin reduced the sensitivity of the enzyme to inhibition by Ca2+. Trifluoperazine (100 microM) reversed both the calmodulin-dependent stimulation of cyclase activity and the calmodulin-induced decrease in sensitivity to the inhibitory actions of Ca2+. Trifluoperazine alone shifted the curve describing Ca2+ inhibition of cyclase activity to the left. The value of Ca2+ for one-half-maximal inhibition decreased from 2.9 to 1.2 microM. The trifluoperazine-induced shift was reversed by exogenous calmodulin. These data suggest: 1) Ca2+, at physiological concentrations, can stimulate as well as inhibit smooth muscle adenylate cyclase activity; 2) the stimulation of adenylate cyclase activity is mediated by calmodulin; 3) the Ca2+-calmodulin complex reduces the sensitivity of smooth muscle adenylate cyclase to the inhibitory actions of Ca2+; and 4) the level of calmodulin associated with smooth muscle adenylate cyclase may modulate the response (both stimulatory and inhibitory) of the enzyme to Ca2+.

Adenylyl Cyclases↗
PubMed1993

Calcium-dependent changes in structure of calmodulin with substance P.

Solution x-ray scattering using synchrotron radiation as an x-ray source has been used to study the solution structure of calmodulin complexed with substance P, a undecapeptide neurotransmitter. The x-ray data indicate that the complex has a compact globular structure, the formation of which is dependent upon the binding of Ca2+ to calmodulin. In the Ca(2+)-saturated condition, the radius of gyration of complexed calmodulin was 4.2 A smaller than that of uncomplexed calmodulin. The Ca(2+)-dependent change in radius of gyration of calmodulin with substance P is complete by the third and fourth Ca2+ binding. The behavior of the Guinier plot at small-to-moderate angles for uncomplexed calmodulin corresponds to a dumbbell shape. The Guinier plot for complexed calmodulin, however, corresponds to a non-dumbbell shape. The susceptibility of calmodulin to proteolytic attack with trypsin was used to examine the nature of the calmodulin complexed with substance P. In the presence of equimolar substance P, the first and second Ca2+ binding to calmodulin was enough to form a trypsin-resistant complex. These biochemical and x-ray data suggest that the binding of substance P to calmodulin is completed when the C-terminal half of calmodulin is occupied by Ca2+, while a significant structural change of calmodulin in the complex is still induced by successive Ca2+ occupancy on the N-terminal half of this molecule.

Amino Acid Sequence↗
PubMed1993

A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis.

Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.

Amino Acid Sequence↗
PubMed1983

Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare).

We report here that calmodulin isolated from the monocotyledon barley is indistinguishable by a variety of criteria from calmodulin isolated from the dicotyledon spinach. In contrast to previous reports, we find that barley (Hordeum vulgare) calmodulin has an amino acid composition similar to that of vertebrate and spinach calmodulins, including the presence of a single trimethyllysinyl residue, and that barley calmodulin quantitatively activates cyclic nucleotide phosphodiesterase. Furthermore, spinach and barley calmodulins are similar in terms of tryptic peptide maps and immunoreactivity with various antisera that differ in their molecular specificities for calmodulins. Limited amino acid sequence analysis demonstrates that the region around the single histidinyl and trimethyllysinyl residues is identical among barley, spinach, and vertebrate calmodulins and that barley calmodulin, like spinach calmodulin, has a novel glutamine residue at position 96. We conclude that calmodulin is highly conserved among higher plants and that detailed sequence analysis is required before significant differences, if any, can be assigned to barley or other higher plant calmodulins. These studies suggest that calmodulin's fundamental importance to the eukaryotic cell may have been established prior to the evolutionary emergence of higher plants.

Journal Article↗
PubMed1994

Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body.

NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.

Alleles↗
PubMed1995

Studies of calcineurin-calmodulin interaction: probing the role of arginine residues using peptidylarginine deiminase.

We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis shows that only 3 of 34 arginines in calcineurin were deiminated; citrulline seems to be localized only in the calcineurin A (CaN A) subunit. Upon incubation with deiminase, the Mn2+/calmodulin-stimulated phosphatase activity decreases to 20-40% of the original activity within 1 h. However, the reduction in enzyme activity is fully protected by addition of calmodulin to the deimination reaction, and only 1.5 mol citrulline/mol calcineurin is found in this case. Removal of the calmodulin binding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested native calcineurin and also results in the loss of all citrulline residues. The calmodulin activation curve of the deiminated enzyme is significantly shifted; the calculated apparent Kact using native calmodulin is 15-fold higher than that of native calcineurin while the apparent Kact using a fluorescent derivative of calmodulin, dansyl-calmodulin, is 10-fold higher. However, the Vm of deiminated calcineurin is similar to that of native if highly elevated levels of calmodulin are used to activate the modified calcineurin. To determine directly if the binding of calmodulin to calcineurin is affected upon deimination, fluorescence titrations using dansyl-calmodulin were performed. The Kd of deiminated calcineurin determined from these titrations is 10-fold higher than that of unmodified calcineurin, indicating that calmodulin binding is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calmodulin binding site of the CaN A subunit.

Animals↗
PubMed1990

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins.

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.

Animals↗
PubMed2000

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site.

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.

Apoproteins↗
PubMed1982

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Preparation of azidocalmodulin: a photoaffinity label for calmodulin-binding proteins.

A photoaffinity label for calmodulin-binding proteins was prepared from 125I-labeled calmodulin (125I-calmodulin) and methyl-4-azidobenzimidate. Azidocalmodulin containing one azido group per calmodulin retained its ability to stimulate the CA2+-sensitive phosphodiesterase purified from bovine heart muscle. The concentrations of calmodulin and azidocalmodulin required for half-maximal stimulation of phosphodiesterase activity were 170 and 230 pM, respectively. Azido-125I-calmodulin was used to photoaffinity label troponin I, myosin light chain kinase, and the Ca2+-sensitive phosphodiesterase. Formation of crosslinked complexes required the presence of Ca2+ or Mn2+ and was inhibited by excess unmodified calmodulin. The calmodulin-binding subunits all formed 1:1 complexes with calmodulin, and the molecular weights of the crosslinked products obtained with troponin I, the phosphodiesterase, and myosin light chain kinase were 43,000, 79,000, and 116,000, respectively. Photolysis experiments using azido-125I-calmodulin and bovine cerebral cortex membranes or detergent-solubilized membranes resulted in formation of a limited number of specifically labeled polypeptides. Azido-calmodulin appears to be an appropriate photoaffinity label for the identification and characterization of calmodulin-binding subunits.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha.

cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase alpha (PTPalpha) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPalpha-D2 in a Ca(2+)-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPalpha, to the two tandem catalytic domains (D1D2) of PTPalpha, nor to the closely related D2 domain of PTPepsilon. Calmodulin bound to PTPalpha-D2 with high affinity, exhibiting a K(D) approximately 3 nm. The calmodulin-binding site was localized to amino acids 520-538 in the N-terminal region of D2. Site-directed mutagenesis showed that Lys-521 and Asn-534 were required for optimum calmodulin binding and that restoration of these amino acids to the counterpart PTPepsilon sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPalpha-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) approximately 340 nm), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPalpha-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPalpha and PTPalpha lacking only D1, but not full-length PTPepsilon, bound to calmodulin beads in the presence of Ca(2+). Also, PTPalpha was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPalpha in vivo but not with PTPalpha-D1D2 in vitro, highlighting a potential conformational difference between these forms of the tandem catalytic domains. The above findings suggest that calmodulin is a possible specific modulator of PTPalpha-D2 and, via D2, of PTPalpha.

Amino Acid Sequence↗
PubMed1984

Adrenal chromaffin cell calmodulin: its subcellular distribution and binding to chromaffin granule membrane proteins.

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.

Adrenal Medulla↗
PubMed1988

A similar calmodulin-binding protein expressed in chromaffin, synaptic, and neurohypophyseal secretory vesicles.

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.

Adrenal Glands↗