Functional conformations of calmodulin: I. Preparation and characterization of a conformational specific anti-bovine calmodulin monoclonal antibody (original) (raw)
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Journal of Biological Chemistry, 1987
Melittin is a 26-amino acid amphipathic peptide which binds to calmodulin in a calcium-dependent manner. The utility of melittin as a peptide replica of the calmodulin-binding region of calmodulin acceptor proteins (CaMBPs) was investigated. Antibody against melittin was raised and purified by antigen affinity chromatography. Interaction of the antibody with CaMBPs was initially suggested by the ability of antimelittin-Sepharose, but not nonimmune IgG-Sepharose, to bind calmodulin-dependent cyclic AMP phosphodiesterase. Direct interaction of melittin antibody with the calmodulin-binding domain of acceptor proteins was demonstrated by quantitative inhibition of calmodulin binding to the purified CaMBPs, myosin light chain kinase, and eel electric organ CaMBP,,. These results indicate that melittin antibody identifies regions of structural similarity between calmodulin acceptor proteins, and this region includes a common calmodulin-binding domain. Calmodulin, a ubiquitous calcium receptor protein, mediates a number of physiological processes including glycogen and cyclic nucleotide metabolism, motility, and secretion (1). Calcium signals transmitted via calmodulin occur through tissue-specific complements of individual calmodulin-binding proteins (CaMBPs).' CaMBPs found in brain include CAMP phosphodiesterase (2, 3), adenylate cyclase (4), protein kinases (5), calcineurin (6), and fodrin (7), as well as additional calmodulin-binding polypeptides as yet functionally undefined (8). In contrast, skeletal muscle and smooth muscle possess as major acceptor proteins, respectively, glycogen phosphorylase kinase (9) and myosin light chain kinase (10). Calmodulin binds to its various acceptor proteins through an amphiphilic interaction (11) which is dependent on the exposure of a calcium-induced hydrophobic region (12-14). Since calmodulin is structurally and functionally conserved throughout all eucaryotes (15-17), it would be anticipated that calmodulin-binding proteins contain a common recognition site, complementary to the site on calmodulin. The search for calmodulin inhibitors of greater specificity and affinity than hydrophobic drugs (18) has led several investigators to focus on the identification of effective, naturally occurring inhibitor peptides of known chemical struc
On the mechanism of interaction between calmodulin and calmodulin-dependent proteins
Biochemistry and Cell Biology, 1983
- On the mechanism of interaction between calmodulin and calmodulin-dependent proteins. Can. J. Biochem. Cell Biol. 61. 91 1-920 Molecular and kinetic studies of the interaction between calmodulin and calmodulin-dependent proteins have been reviewed. Several calmodulin-dependent proteins have been purified to homogeneity and characterized in terms of subunit structure in recent years. The results indicate that these proteins do not contain a common subunit as the basis of calmodulin binding. A monoclonal antibody capable of interacting with several calmodulin-dependent proteins has been obtained, suggesting that these proteins contain common structure. It seems that hybridoma technology may be used for probing calmodulin-binding domains in the calmodulin-dependent proteins. Using a fluorescent-labelled cyclic nucleotide phosphodiesterase, the interaction between calmodulin and the enzyme in the absence of ~a " can be demonstrated, and the equilibrium constant of the reaction can be determined. The study further defines the multiple interactions in the activation of the cyclic nucleotide phosphodiesterase by ~a " and calmodulin. Previous kinetic results along with the present results are summarized and used to elucidate the regulatory significance of the multiple ca2+-binding of calmodulin. 1983) On the mechanism of interaction between calmodulin and calmodulin-dependent proteins. Can. J. Biochem. Cell Biol. 61, 9 1 1-920 Nous rCvisons les Ctudes molCculaires et cinktiques de l'interaction entre la calmoduline et les protCines ddpandantes de la calmoduline. Au cours des derni&res annkes, plusieurs protkines ddpendantes de la calmoduline ont Ct C purifikes jusqu'h homogCnCitC et caractCrisCes en temes de structure sous-unitaire. Les rksultats montrent que ces protCines ne contiennent pas de sous-unit6 commune qui serait a la base de la liaison h la calmoduline. On a obtenu un anticorps monoclonal capable de rdagir avec plusieurs protCines ddpndantes de la calmoduline; c'est 18 un indice que ces protCines contiennent une structure commune.
Proceedings of the National Academy of Sciences
Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by -40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and
Journal of Immunological Methods, 1991
A simple linearization procedure has been developed to determine the apparent dissociation constant of the interaction between antigen and antibody from the data of indirect, non-competitive enzyme-linked immunosorbent assays (ELISA). Applying this dissociation constant the binding constant of ligands to antigen can be determined and the quantitative evaluation of the competitive ELISA experiments makes it possible to analyse the affinity of antibody to antigen on the surface and in solution.
Analytical Biochemistry, 2009
This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ($80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC 50 values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.
Journal of Biological Chemistry
The Ca2+-dependent association of &endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(8aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the t-amino groups of the seven lysyl residues on calmodulin by ['Hlacetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where &endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-med1ated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (
Biochemical Journal, 1992
An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N-and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluoperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.
Calmodulin-binding proteins of
Biochemical and Biophysical Research Communications, 1990
Calcium is involved in the regulation of a variety of cellular processes in eucaryotic cells. For example, changes in intracellular Ca 2+ levels regulate the progression through specific stages of the cell cycle (1). Ca2+ is also involved in protein secretion and in signaling by various extracellular ligands. These processes appear to be mediated by Ca2+ fluxes within the cell. Calmodulin is a highly conserved, major Ca2+-binding protein that is found in all eucaryotic Cop?tright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Immunoelectron microscopic localization of calmodulin with protein A-gold technique
Acta Histochemica et Cytochemica, 1984
The localization of calmodulin in guinea pig testis and spermatozoa was determined by both immunoperoxidase labeling and immunocolloidal-gold methods on the electron microscopic level. In spermatids, reaction deposits were scattered all over the cytoplasm, but hardly recognized in acrosome vesicle and Golgi complex. In the neck portion of elongated spermatids, anti-calmodulin immunoreactive sites were diffusely localized in cytoplasm, and also recognized along the axial filaments and near the connecting pieces. Mature spermatozoa exhibited the following distinct regions of anti-calmodulin immunoreactive sites; the cytoplasm between plasma and outer acrosomal membranes, between nuclear envelope and inner acrosome membrane, along the axial filaments, and outside of fibrous sheath. These findings suggest that calmodulin may play a role in both acrosome reaction and tail movement. Calmodulin, ubiquitous in eukaryotic systems, has been implicated as a multifunctional regulatory mediator of calcium in the cytoplasm of cells (5, 17, 23, 24). By microscopic and biochemical methods, sites with increased amounts of calmodulin have been demonstrated in mitotic apparatus (13, 25-27), ciliary structures (15, 19), the microvillar structures of the intestinal brush border (4, 6, 8), and in the synapses (7, 14, 20). The acrosome reaction of capacitated spermatozoa was induced in the presence of extracellular calcium ion (29), and relatively high levels of calmodulin were recognized in spermatozoa from mammals and sea urchins (9). By immunofluorescence technique, Jones et al. (10) observed that calmodulin was exclusively located in the head cap of spematozoa. The present study was undertaken to show the detailed distribution of calmodulin at the higher resolution level of electron microscopy in both guinea pig testis and spermatozoa, and to elucidate the biological role of calmodulin in spermatozoa. A part of this study has been reported previously (30). MATERIALS AND METHODS Calmodulin was purified from rat testis by the method of Kakiuchi et al. (11).