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PubMed1980

Positive cooperative binding of calcium to bovine brain calmodulin.

Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.

Animals↗
PubMed1980

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin.

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

Animals↗
PubMed2005

Kinetic analysis of the calmodulin-binding region of the plasma membrane calcium pump isoform 4b.

The sequence L(1086)RRGQILWFRGLNRIQTQIKVVKAFHSS(1113) (peptide C28) is responsible for calmodulin binding to PMCA4b. In this work, peptides following the above sequence were progressively shortened either at the N-terminus (C28NDelta3, C28NDelta5, or C28NDelta6) or at the C-terminus (C20, C22, C23, and C25). Competitive inhibition of PMCA activity was used to measure apparent dissociation constants of the complexes between calmodulin and C28 or progressively shortened peptides. Additionally, equilibrium titrations were used to measure the apparent dissociation constants of the various peptides with TA-calmodulin by changes in TA-calmodulin fluorescence and Trp fluorescence of the peptides. At the N-terminus, deletion of five residues did not change calmodulin affinity, but deletion of six residues resulted in a 5-fold decrease in affinity. There were no major differences in the time course of TA-CaM binding, but C28NDelta6 exhibited a different time course of Trp fluorescence change. At the C-terminus, deletion of five residues (C23) or more resulted in a net increase in fluorescence of TA-CaM upon binding, while longer peptides (C25 and C28) produced both a transient increase and a net decrease in the fluorescence of TA-CaM. Global regression analysis revealed that binding of TA-CaM to the C23 peptide could be fit by a two-step model, while longer peptides required three-step models for adequate fitting. TA-calmodulin dissociated rapidly from C23, C22, and C20, resulting in a marked increase in apparent K(d). Thus, the sequence I(1091)LWFRGLNRIQTQIKVVKAF(1110) (C25NDelta5) is required to reproduce the calmodulin-binding properties of C28. When F(1110) was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V(1107) to A produced a smaller effect, suggesting that F(1110), rather than V(1107), is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.

Amino Acid Sequence↗
PubMed1996

Distinct molecular recognition of calmodulin-binding sites in the neuronal and macrophage nitric oxide synthases: a surface plasmon resonance study.

The neuronal nitric oxide synthase and the macrophage nitric oxide synthase are differently regulated by Ca2+/calmodulin. We investigated the dynamics of calmodulin binding to the putative calmodulin-binding sites in both nitric oxide synthases. Peptides derived from the putative calmodulin-binding sites were synthesized and immobilized to a dextran layer of a biosensor chip. Complex formation of calmodulin and the peptides was monitored by surface plasmon resonance spectroscopy and recorded as sensorgrams. We determined a dissociation constant KD of 5.0 x 10(-9) M for the neuronal nitric oxide synthase and calmodulin. The association rate constant and the dissociation rate constant were ka = 1.58 x 10(5) M-1 s-1 and kd = 7.87 x 10(-4) s-1, respectively. Sensorgrams obtained with the macrophage nitric oxide synthase peptide were remarkably different. Calmodulin, once bound to the peptide, did not dissociate. Association of calmodulin to the peptide occurred with the same rate constants (ka = 3 x 10(4) M-1 s-1) regardless of the presence or absence of Ca2+. The affinity was in the subnanomolar range (KD) < 0.1 x 10(-9) M). We conclude that the extremely tight binding of calmodulin to the NOS-II is solely controlled by the calmodulin-binding segment and not by other parts of the protein.

Animals↗
PubMed1996

Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin.

EPR spectroscopy was used to examine protein-protein interactions between calmodulin and spin-labeled peptides based on the protein kinase C substrate domains of the myristoylated alanine rich C kinase substrate (MARCKS) and neuromodulin. When bound to calmodulin, the C- and N-terminal ends of a 25 residue MARCKS derived peptide exhibited large amplitude motion on the nanosecond time scale and were accessible to paramagnetic agents in aqueous solution. However, residues 5-23 were highly protected and in contact with side chains from calmodulin. These data are consistent with an alpha-helical configuration for this segment of MARCKS and with structures that have been obtained for other calmodulin-substrate complexes. For the 17 residue neuromodulin derived peptide, which is Ca2+ independent in its binding to calmodulin, oxygen collision rates demonstrate that one helical face of this peptide interacts strongly with calmodulin. The data are consistent with an interaction of this face specifically with the C-terminal lobe of calmodulin, where this lobe is either in an "open" or "semiopen" configuration. The EPR data also indicate that the N-terminal lobe of calmodulin is in contact with the peptide, but that this lobe is not as strongly associated with the peptide target. Overall, the binding pocket for neuromodulin appears to be less compact and more dynamic than that formed by MARCKS. This behavior has not previously been seen for calmodulin substrates, and it may play a role in the Ca2+ independent binding of this class of substrates. This work demonstrates the utility of EPR spectroscopy to define protein-protein interactions; in addition, oxygen collision frequencies obtained at buried sites appear to provide information on the conformational dynamics of proteins.

Amino Acid Sequence↗
PubMed1998

Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and spectroscopic studies.

The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 A resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3"-(beta-chloroethyl)-2",4"-dioxo-3, 5"-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.

Animals↗
PubMed1997

Stellettamide-A, a novel inhibitor of calmodulin, isolated from a marine sponge.

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 microM. 2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 microM)-induced contractions with an IC50 of 46 microM for 3 microM Ca2+ and 105 microM for 10 microM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. 3. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 microM. 4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 microM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. 5. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 microM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 microM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. 6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3-300 microM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. 7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

Animals↗
PubMed1979

Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.

Animals↗
PubMed1980

The preparation of calmodulins from barley (Hordeum sp.) and basidiomycete fungi.

1. Calmodulin-like proteins were purified from the fruiting bodies of higher (basidiomycete) fungi and barley (Hordeum sp.) shoots. 2. These calmodulins have electrophoretic mobilities on 10% (w/v) polyacrylamide gels at pH 8.3 in the presence of 6 M-urea and at pH 8.3 in the presence of 0.1% sodium dodecyl sulphate similar to that of bovine brain calmodulin. They interacted with rabbit skeletal-muscle troponin I in the presence of Ca2+. 3. Barley and fungal calmodulins activated myosin light-chain kinase and phosphodiesterase in the presence of Ca2+, although the amounts needed were at least an order of magnitude greater than is required to produce the same effect with mammalian calmodulin. 4. Amino acid analyses indicated a number of differences from the mammalian protein, most notably the absence of trimethyl-lysine. 5. By using 125I-labelled calmodulin, a small amount of calmodulin-binding protein was detected in homogenates of barley and fungi. 6. No protein corresponding to calmodulin could be found in Escherichia coli or yeast, although a relatively high concentration of a protein that bound calmodulin was detected in E. coli by this technique.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2007

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization.

Ca2+ signalling to the nucleus is thought to occur by calmodulin entry into the nucleus where calmodulin has many functions. In the present study we have investigated the role of Ca2+ and the N- and C-terminal lobes of calmodulin in its subnuclear targeting by using fluorescently labelled calmodulin and its mutants and confocal microscopy. Our data show, first, that Ca2+ stimulation induces a reorganization of subnuclear structures to which apo-calmodulin can bind. Secondly, Ca2+-independent association of the C-terminal lobe is seen with subnuclear structures such as chromatin, the nuclear envelope and the nucleoli. Thirdly, Ca2+-dependent accumulation of both calmodulin and the C-terminal calmodulin lobe occurs in the nucleoli. The N-terminal lobe of calmodulin does not show significant binding to subnuclear structures although, similarly to the C-terminal lobe, it accumulates in the nucleoplasm of wheat germ agglutinin-blocked nuclei suggesting that a facilitated nuclear export mechanism exists for calmodulin.

Active Transport, Cell Nucleus↗
PubMed1982

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The effects of Ca2+-calmodulin on adenylate cyclase activity in EGTA-washed, 27000 g particulate fractions of mouse and rat pancreatic islets were studied. Ca2+ (10 microM)-calmodulin (1 microM) stimulated adenylate cyclase activity 53.1 +/- 5.2 (N = 6)% in the particulate fraction of rat islets. Trifluoperazine (50 microM), a specific inhibitor of calmodulin, inhibited the Ca2+-calmodulin activation of the adenylate cyclase activity of this fraction of rat islets. These results confirm previous reports dealing with Ca2+-Calmodulin and rat islet adenylate cyclase [Valverde, Vandermeers. Anjaneyulu & Malaisse (1979) Science 206, 225-227; Sharp, Wiedenkeller, Kaelin, Siegel & Wollheim (1980) Diabetes 29, 74-77]. In contrast, however, Ca2+ (1-100 microM)-calmodulin (1-10 microM) did not stimulate the adenylate cyclase activity in the EGTA-washed particulate fraction of mouse islets, and trifluoperazine (50 microM) did not inhibit the adenylate cyclase activity of this fraction of mouse islets, although some remaining calmodulin [0.18 +/- 0.05 (n = 3) microgram/mg of protein] could be demonstrated. GTP (10 microM) enhanced islet adenylate cyclase activity considerably, but did not confer any sensitivity towards Ca2+-calmodulin on mouse islet adenylate cyclase. The results question the role of calmodulin in the Ca2+-dependent rise in cyclic AMP evoked by glucose in pancreatic islets.

Adenylyl Cyclases↗
PubMed1984

Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma.

Mouse B16 melanoma extracts of both cultured cells and tumour tissue contain cyclic AMP phosphodiesterase activity, with 95% present in the soluble fraction. Although activation of the enzyme by added calmodulin did not occur, it was found that endogenous calmodulin was present at a level sufficient to activate fully the enzyme. The ability of Ca-calmodulin to stimulate cyclic AMP phosphodiesterase in this tissue was shown by the inhibitory effect of N-(6-aminohexyl)-5-chloronaphthalenesulphonamide (W7), a known calmodulin antagonist; by the activation of the enzyme with exogenous calmodulin observed in supernatants depleted of endogenous calmodulin by passage over fluphenazine-Sepharose 6B in the presence of Ca2+; by the Ca-dependent binding of the enzyme to calmodulin-agarose and its activation by Ca-calmodulin after elution from the column with EGTA-containing buffer. It was calculated that about 50% of the total cyclic AMP phosphodiesterase activity was calmodulin-activated in this tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes.

Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.

Animals↗
PubMed1981

Interactions of calmodulin with coated vesicles from brain.

Coated vesicles purified in the presence of calcium are enriched approximately 7-fold in calmodulin content relative to standard preparations isolated in the absence of free calcium. Radioiodinated calmodulin binds specifically to coated vesicles in vitro. Binding is saturable (Kd, 10 nM) and calcium dependent. Half-maximal binding occurs at 2.4 microM free Ca2+ whereas up to 1.2 mM Mg2+ has no effect on binding. Troponin C, a protein homologous to calmodulin, competes with binding of 125I-labeled calmodulin with 1/30th the affinity of native calmodulin. Chromatography of 2 M urea-solubilized coated vesicles on a calmodulin-Sepharose column demonstrated a Ca2+-dependent interaction of coated vesicle proteins and calmodulin. The properties of calmodulin binding to coated vesicles are comparable to those of calmodulin activities in other systems.

Animals↗
PubMed1983

Engineering of site-directed antisera against vertebrate calmodulin by using synthetic peptide immunogens containing an immunoreactive site.

Site-directed antisera against vertebrate calmodulin were elicited in rabbits by injection of a synthetic immunogen containing the pentadecapeptide Gly-Gln-Val-Asn-Tyr-Glu-Glu-Phe-Val-Gln-Met-Met-Thr-Ala-Lys-OH, which corresponds to residues 134-148 of vertebrate calmodulin. A major immunoreactive region (residues 127-144) of calmodulin is found in the COOH-terminal structural domain and an immunoreactive site for one antiserum is contained in the heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2, which corresponds to residues 137-143 of vertebrate calmodulin. This immunoreactive heptapeptide was conjugated to a carrier protein by adding a cysteine residue to the NH2 terminus of the peptide and coupling the Cys-heptapeptide to the carrier through the thiol group of the cysteine residue. Injection of this Cys-heptapeptide-protein conjugate into rabbits yielded antisera that react with the heptapeptide but not with native calmodulin. Thus, the immunoreactive heptapeptide that is exposed on the surface of calmodulin is immunogenic, but it is not sufficient to elicit antibodies that react with native calmodulin. However, when the Cys-pentadecapeptide corresponding to residues 134-148 and containing the immunoreactive heptapeptide sequence was conjugated to a carrier protein and injected into rabbits, antisera were elicited that react with the intact calmodulin molecule. The affinities and specificities of these antisera for calmodulin are similar to those of antisera elicited by injection of the intact protein and are sufficient for their use in radioimmunoassays. These results indicate that the successful engineering of site-directed antisera against proteins by using synthetic peptide immunogens may require an appropriate intramolecular environment that allows the peptide region to closely approximate the spatial orientation it adopts in the intact protein.

Amino Acid Sequence↗
PubMed1989

In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115.

The Paramecium tetraurelia mutants termed pantophobiacs have altered behavior due to perturbed calcium activation of ion channel activity. The calmodulin from pantophobiac A1 (pntA1) was shown in previous studies to have a single amino acid change at residue 101 that is selective in its effects on activity. This change has no effect on posttranslational modifications. However, the calmodulin from the phenotypically related mutant pantophobiac A2 (pntA2) has a threonine residue at position 136, in the fourth calcium-binding domain, instead of an isoleucine or valine like all other calmodulins. This region of the calmodulin structure is within 4 A of a complementary hydrophobic structure in the third calcium-binding domain, raising the possibility of a perturbation of interdomain interactions in the pntA2 mutant. This possibility is supported by the heterogenous methylation state of lysine-115 in the pntA2 calmodulin. This lysine residue, located in the peptide connecting calcium-binding domains three and four, is fully trimethylated in the wild-type and pntA1 calmodulins. The functional selectivity of these structural changes is demonstrated by the conservation of calmodulin activator activity with a calmodulin-regulated protein kinase that has been used as a standard of comparison. Overall, these results indicate the degree to which the calmodulin can be mutated in vivo without being lethal to the organism, and they provide genetic evidence suggesting that the post-translational methylation state of residue 115 requires the appropriate conformation in addition to the local amino acid sequence.

Amino Acid Sequence↗
PubMed1996

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Animals↗
PubMed1995

A flagellar calmodulin gene of Naegleria, coexpressed during differentiation with flagellar tubulin genes, shares DNA, RNA, and encoded protein sequence elements.

Two calmodulins are synthesized during differentiation of Naegleria gruberi from amoebae to flagellates; one remains in the cell body and the other becomes localized in the flagella. The single, intronless, expressed gene for flagellar calmodulin has been cloned and sequenced. The encoded protein is a typical calmodulin with four putative calcium-binding domains, but it has an amino-terminal extension of 10 divergent amino acids preceding conserved calmodulin residue 4. The transcripts encoding flagellar calmodulin and flagellate cell body calmodulin are clearly divergent. Expression of the flagellar calmodulin gene is differentiation-specific; its mRNA appears and then disappears concurrently with those encoding flagellar alpha- and beta-tubulin. Three provocative sequence elements are shared among these unrelated coexpressed genes: (i) a palindromic DNA sequence element is found in duplicate or triplicate upstream to each transcribed region; (ii) a perfect 12-nucleotide match is found near the AUG start codon of flagellar calmodulin and alpha-tubulin; and (iii) the novel amino-terminal extension of flagellar calmodulin contains a 5-amino-acid element similar to the amino terminus of flagellar alpha-tubulin. These shared sequence elements are proposed to have roles in differentiation, possibly in regulation of transcription, mRNA stability, and localization of these proteins to flagella.

Amino Acid Sequence↗