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PubMed1982

Affinity labeling of calmodulin-binding components in canine cardiac sarcoplasmic reticulum.

Canine cardiac sarcoplasmic reticulum vesicles have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated a 125I-containing major product of Mr = 40,000, and a minor component of Mr = 120,000. This latter component probably represents a 1:1 complex between the 100,000-dalton Ca2+-ATPase protein and 125I-calmodulin. When cross-linked samples, solubilized in sodium dodecyl sulfate, were boiled for 2 min, the radioactivity associated with the 40,000-dalton component decreased while that associated with components of 26,000 and 28,000 daltons increased. When these boiled samples were stored at -70 degrees C for 1 week, the radioactivity associated with the 26,000- and 28,000-dalton components decreased whereas that associated with the 40,000-dalton component was increased. This suggests that the 40,000-dalton component represents a 1:1 cross-link between the 23,000-dalton form of phospholamban and 125I-calmodulin. The 26,000- and 28,000-dalton cross-linked components probably represent 1:1 cross-links between 125I-calmodulin and the 8,000- and 11,000-dalton subunits, respectively, of phospholamban. A 32P-containing, 40,000-dalton component was formed when dithiobis(succinimidyl propionate) was added to sarcoplasmic reticulum vesicles, phosphorylated in the presence of [gamma-32P]ATP and 3 microM calmodulin. This confirms that the 40,000-dalton affinity-labeled component is a 1:1 cross-link between phospholamban and calmodulin. We propose that phospholamban is the endogenous receptor for calmodulin in cardiac sarcoplasmic reticulum membranes. However, it is unlikely that phospholamban is the endogenous calmodulin-dependent protein kinase in this membrane.

Animals↗
PubMed1995

A calmodulin-binding sequence in the C-terminus of human cardiac titin kinase.

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the substrate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt alpha-helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.

Amino Acid Sequence↗
PubMed1984

Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases.

Cilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5-min extraction was generally insensitive to added calmodulin, whereas that obtained by 20-hr extraction of the 5-min extracted axonemes was activated by calmodulin, the activation being much larger in the "light" 14S fractions than in the "heavy" fractions. The 30S dynein ATPase obtained by a 5-min extraction was generally activated over 1.6-fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3-fold. After further purification of the 5-min extracted 30S dynein and of the 5-min to 20-hr-extracted 14S dynein on DEAE-Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro-9-[3-(2-hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms that are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.

Adenine↗
PubMed1984

Prereplicative changes in the soluble calmodulin of isoproterenol-activated rat parotid glands.

Cells of rat parotid glands were maximally stimulated to initiate DNA synthesis by injecting into the animal a single dose of 25 to 150 mg of isoproterenol/kg of body weight. During the 18- to 21-hr prereplicative period following injection of the highest dose of the drug, there were two predominant and transient redistributions of calmodulin from the bound to the soluble form, which tripled the level of soluble calmodulin at 3 hr and again at 18 hr just before the initiation of DNA synthesis. A small (50%) increase in total calmodulin was observed only during the early (3-h) prereplicative surge of soluble calmodulin. The late, pre-DNA-synthetic surge of soluble calmodulin and the initiation of DNA synthesis were both prevented in rats that lacked their parathyroid-thyroid gland complex and had been hypocalcemic for 48 or 72 hr. Unlike the effect of high doses of isoproterenol, low doses (e.g., 25 mg/kg body weight) of the beta-adrenergic drug could maximally stimulate DNA synthetic activity without the later pre-DNA-synthetic surge of soluble calmodulin, suggesting that any apparent correlation between the level of calmodulin and DNA synthesis may be spurious and that an actual increase in the level of soluble calmodulin just before the onset of DNA synthesis was not a prerequisite for DNA synthetic activity in parotid cells.

Animals↗
PubMed1987

Immunohistochemical study of calmodulin in developing mouse testis.

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.

Aging↗
PubMed1992

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.

Calcium↗
PubMed1989

The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation.

Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein.

Affinity Labels↗
PubMed1996

Expression of HIV-1 envelope glycoprotein alters cellular calmodulin.

Removal of parts of a known calmodulin binding site at the C-terminus of HIV-1 envelope glycoprotein, gp160, can result in diminished infectivity. We investigated whether expression of full length gp160 would result in changes in intracellular calmodulin compared to expression of gp160 truncated to remove both known calmodulin binding sites. Both Western and Northern blots demonstrated that expression of gp160 led to increased calmodulin when compared to expression of truncated gp160. The induced calmodulin was associated preferentially with a particulate subcellular fraction. Confocal immunomicroscopy confirmed the increase in calmodulin and also showed that there was enhanced colocalization of calmodulin with gp160. Understanding of the role of calmodulin in the viral life-cycle may lead to new therapeutics.

Binding Sites↗
PubMed2001

Interaction of plant protein Ser/Thr phosphatase PP7 with calmodulin.

We have recently identified PP7, a novel group of plant protein Ser/Thr phosphatases, and hypothesized that PP7 may possess a calmodulin-binding site. To test this hypothesis, we assessed the effect of calmodulin on the activity of recombinant Arabidopsis thaliana PP7 and directly tested interaction between PP7 and calmodulin using surface plasmon resonance. Calmodulin exerted a moderate inhibitory effect on the phosphatase activity of PP7 with submicromolar affinity. PP7 specifically interacted with immobilized calmodulin (but not with recoverin, another EF hand Ca(2+)-binding protein) in a strictly Ca(2+)-dependent manner with nanomolar affinity. Deletion of an insert in the catalytic domain of PP7, predicted to function as a calmodulin-binding site, greatly decreased PP7 binding to calmodulin. These findings provide the first evidence for a plant protein phosphatase directly interacting with calmodulin and indicate that PP7 might be regulated by Ca(2+) levels in vivo.

Arabidopsis↗
PubMed1997

Energetics of target peptide recognition by calmodulin: a calorimetric study.

Calmodulin is a small protein involved in the regulation of a wide variety of intracellular processes. The cooperative binding of Ca2+ to calmodulin's two Ca2+ binding domains induces conformational changes which allow calmodulin to activate specific target enzymes. The association of calmodulin with a peptide corresponding to the calmodulin binding site of rabbit smooth muscle myosin light chain kinase (smMLCKp) was studied using isothermal titration microcalorimetry. The dependence of the binding energetics on temperature, pH, Ca2+ concentration, and NaCl concentration were determined. It is found that the binding of calmodulin to smMLCKp proceeds with negative changes in enthalpy (deltaH), heat capacity (deltaCp), and entropy (deltaS) near room temperature, indicating that it is an enthalpically driven process that is entropically unfavorable. From these results it is concluded that the hydrophobic effect, an entropic effect which favors the removal of non-polar protein groups from water, is not a major driving force in calmodulin-smMLCKp recognition. Although a large number of non-polar side-chains are buried upon binding, these stabilize the complex primarily by forming tightly packed van der Waals interactions with one another. Binding at acidic pH was studied in order to assess the contribution of electrostatic interactions to binding. It is found that moving to acidic pH results in a large decrease in the Gibbs free energy of binding but no change in the enthalpy, indicating that electrostatic interactions contribute only entropically to the binding energetics. The accessible surface area and atomic packing density of the calmodulin-smMLCKp crystal structure are analyzed, and the results discussed in relation to the experimental data.

Amino Acid Sequence↗
PubMed2000

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

Amino Acid Sequence↗
PubMed2001

Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Animals↗
PubMed1983

Lead and other metals can substitute for Ca2+ in calmodulin.

We have studied the interaction between some heavy metal ions, as compared with earth alkali ions, and calmodulin, a tissue protein which binds Ca2+ and mediates some of its effects. 1. Calmodulin dependent phosphodiesterase was activated with Pb2+, Ca2+, Sr2+, Ba2+, and Cd2+ (EC50 about 0.8 microM). The maximal activation achieved decreases in the order given. Hg2+, Sn2+, Fe2+, Cu2+, Ni2+, Bi3+, and Sb3+ up to 20 microM did not activate. 2. Pb2+ can replace Ca2+ with respect to the calmodulin-dependent phosphorylation of brain membranes. With high Pb2+ concentrations, phosphorylation was inhibited. 3. Calmodulin binding to brain membranes was enhanced with concentrations below 10(-4)M in the following order: Pb2+ greater than or equal to Ca2+ approximately Sr2+ greater than Cd2+ greater than Mn2+ greater than Ba2+. In contrast Mg2+, Hg2+, Sn2+, Fe2+, Ni2+, Co2+, and Cu2+ triggered, if at all, a non-saturable binding of calmodulin. 4. In the flow-dialysis, other ions competed with 45Ca2+ binding to calmodulin in the following order: Pb2+ approximately Ca2+ greater than Mn2+, Ba2+, Cd2+, Sr2+. Thus among the ions investigated Pb2+ is a fully potent substitute for Ca2+ in every calmodulin-dependent reaction investigated. Cd2+ is always much less potent. The earth alkali ions Sr2+ and Ba2+ take an intermediate position. It remains to be shown whether calmodulin is merely a storage site for Pb2+, or whether the resulting functional changes play a role in Pb2+ poisoning.

Animals↗
PubMed1983

The effect of calmodulin antagonists on amylase release from the rat parotid gland in vitro.

The effect of several calmodulin antagonists on the release of alpha-amylase (EC 3.2.1.1) from rat parotid gland minces was investigated as an approach to determine whether calmodulin has a role in the stimulus-secretion coupling mechanism in this tissue. The phenothiazines, trifluoperazine, chlorpromazine, and thioridazine, failed to inhibit amylase release induced by N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate. All three phenothiazines increased basal amylase release at high concentrations. This release was independent of cellular energy, indicating that the release was probably due to the membrane perturbing properties of these compounds rather than their ability to antagonize calmodulin. R24571, a more potent calmodulin antagonist, also failed to inhibit amylase release induced by N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate but increased basal amylase release. A different calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, partially inhibited release while 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) completely blocked the amylase release induced by the cyclic AMP derivative. However, concentrations of TMB-8 inhibiting amylase release drastically reduced the ATP concentration of rat parotid minces, suggesting that the inhibition of secretion was due to toxic effects of TMB-8 on parotid cells rather than the ability of the compound to antagonize calmodulin. The use of calmodulin antagonists has therefore failed to implicate calmodulin as an intermediate in the stimulus-secretion coupling mechanism of the rat parotid gland.

Adenosine Triphosphate↗
PubMed1987

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts.

The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 microM, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+- and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts.

Animals↗
PubMed1996

Ultrastructural evidence for the lack of co-transport of B-50/GAP-43 and calmodulin in myelinated axons of the regenerating rat sciatic nerve.

Following peripheral nerve injury, neurons respond with synthesis of proteins required for axonal regeneration. Newly synthesized membrane proteins, like B-50/GAP-43, are transported with the fast component of anterograde axonal transport. Structural proteins and calmodulin are transported by the slow component. Since B-50/GAP-43 can bind calmodulin, it has been hypothesised that B-50/GAP-43 may act as a carrier for fast anterograde transport of calmodulin, so that both proteins are delivered rapidly to the distally outgrowing axons ('the fast carrier hypothesis'). We have investigated whether this hypothesis is valid in myelinated axons of the regenerating rat sciatic nerve. Seven days after crush, the nerve was ligated to accumulate fast transported proteins. Nerve pieces were dissected proximal to the ligation and processed for immunofluorescence and quantitative electron microscopy by postembedding single and double immunogold labelling. By light microscopy, we observed a qualitative increase in B-50/GAP-43 immunofluorescence in the axonal element immediately proximal to the nerve ligation (termed 'accumulated') compared to an upstream site (termed 'regenerating') closer to the cell body. The immunofluorescence for calmodulin appeared to be the same at both sites. Using electron microscopy, we observed that organelles had collected at the 'accumulated' site, moreover the density of B-50/GAP-43 immunolabelling was significantly increased compared to the 'regenerating' site, where the axoplasmic structure was undisturbed. The increase in B-50/GAP-43 immunolabelling was largely associated with vesicles. The density of calmodulin immunolabelling was similar at both sites. Approximately 25% of the total B-50/GAP-43 was associated with vesicles of which only 15% also contained labelling for calmodulin. Thus, ligation of the nerve resulted in accumulation of vesicles, including those carrying B-50/GAP-43, largely without calmodulin. Therefore, contrary to 'the fast carrier hypothesis', the bulk of calmodulin is not co-transported with B-50/GAP-43 in myelinated axons of the sciatic nerve.

Animals↗
PubMed1983

Calmodulin and its roles in skeletal muscle function.

The purpose of this review is to describe the importance of calmodulin as a mediator of the effects of calcium ions in living systems, particularly in the process of skeletal muscle contraction. Calmodulin is a low molecular weight, acidic, calcium binding protein which mediates the Ca2+ regulation of a wide range of physiological processes throughout eukaryotic organisms. At low free Ca2+ concentrations, such as exist in resting muscle sarcoplasm, calmodulin exists in the Ca2+-free form in which state it does not generally interact with a target protein. Following an appropriate stimulus, the free Ca2+ concentration rises whereupon Ca2+ binds to calmodulin which undergoes a conformational change enabling it to interact with a target protein(s). The overall result of this protein-protein interaction is a physiological effect, e.g., Ca2+ binding to calmodulin in smooth muscle allows it to interact with and activate myosin light chain kinase which catalyzes the phosphorylation of myosin. This reaction results in contraction of the smooth muscle. Recent studies have implicated calmodulin in the Ca2+ control of three enzymes in skeletal muscle: phosphorylase kinase, myosin light chain kinase and a protein kinase of the sarcoplasmic reticulum. Various classes of drugs, including certain local anaesthetics, have been shown to affect calmodulin-dependent processes. It is likely that the effects of such drugs result from their interaction with calmodulin.

Animals↗
PubMed1983

Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin.

Calmodulin from both animal and plant sources is known to bind a number of hydrophobic compounds with resultant inhibition of calmodulin function. Some of these compounds, including certain phenothiazine and naphthalene sulfonamide derivatives, have been previously shown to be useful in the chromatographic isolation of calmodulin, when covalently linked to a solid support. With the exception of fluphenazine linked to epoxide-activated Sepharose, these resins have the undesirable characteristics of requiring high salt concentrations in the elution buffer for efficient elution of calmodulin, thus decreasing the selectivity for this protein. The synthesis of nine Sepharose-ligand affinity resins is reported. Some of the ligands are newly synthesized naphthalene sulfonamide and phenothiazine derivatives. The synthetic ligands have been coupled to three types of Sepharose: epoxide-activated, CNBr-activated, and carbodiimide-activated. The properties of these resins are reported and their relative abilities to act selectively in the isolation of calmodulin are compared. 2-Trifluoromethyl-10-aminopropyl phenothiazine (TAPP), when linked to epoxide-activated Sepharose, was found to be the most useful for calmodulin isolation in terms of its combined stability, capacity, and ability to select for calmodulin. This resin was found to behave as a true affinity resin. A quantitative evaluation of its affinity behavior was consistent with the presence of two high-affinity Ca2+-dependent phenothiazine binding sites on calmodulin, in apparent agreement with previous reports which involved the use of different methods.

Animals↗