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PubMed1989

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering.

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.

Animals↗
PubMed1988

Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes.

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.

Amino Acid Sequence↗
PubMed1987

Selective calmodulin inhibition toward myosin light chain kinase by a new cerebral circulation improver, Ro 22-4839.

Ro 22-4839, a new cerebral circulation improver, has shown to be a potent calmodulin antagonist toward myosin light chain kinase (MLCK). It inhibited in vitro activity of calmodulin-activated cyclic AMP phosphodiesterase isolated from either bovine heart or brain and ATP-induced superprecipitation of chicken gizzard actomyosin with respective IC50 values of 20 microM, 17 microM, and 2.0 microM. The inhibitory action of Ro 22-4839 on the contractile system of the smooth muscle was demonstrated directly by its inhibition of chicken gizzard MLCK. Ro 22-4839 was found to potently inhibit MLCK with an IC50 value of 3.1 microM but was unable to inhibit the activity of MLCK rendered Ca2+/calmodulin independent by limited tryptic digestion. The inhibition of MLCK induced by Ro 22-4839 was completely overcome by addition of excess calmodulin. In contrast, Ro 22-4839 hardly inhibited calmodulin-activated Ca2+, Mg2+-ATPase from rat erythrocyte membrane or adenylate cyclase from rat brain. Use of hydrophobic fluorescence probes showed that Ro 22-4839 binds to the hydrophobic region of calmodulin like other calmodulin antagonists, trifluoperazine and W-7. However, the precise binding site of Ro 22-4839 to calmodulin is different from those of trifluoperazine and W-7, as suggested from differing IC50 values of these compounds against the probes. We conclude that Ro 22-4839 inhibits calmodulin-activated enzymes, most significantly of MLCK, highly specific to smooth muscle contractile systems by binding to the hydrophobic domain of the calmodulin and inducing its conformational change in the presence of calcium.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase.

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.

Animals↗
PubMed1985

Bovine lens calmodulin. Isolation, partial characterization and calcium-independent binding to lens membrane proteins.

Calmodulin has been isolated from calf lens fiber cells. Like other vertebrate calmodulins lens calmodulin shows a calcium-dependent mobility shift on SDS-polyacrylamide gels and forms immune complexes with antiserum, raised against vertebrate calmodulin. Via the gel overlay technique radioiodinated calmodulin from lens or bovine brain was found to bind to the main intrinsic protein (MIP) and the 17.5 kDa protein of lens fiber membranes in a calcium-independent manner. After proteolytic digestion of lens fiber membranes with trypsin or Staphylococcus aureus V8 protease the calmodulin-binding activity of MIP is retained. This result indicates that the small polypeptide fragment of MIP, which is accessible to proteolytic attack, apparently is not the attachment point for calmodulin. Two additional calmodulin-binding proteins (MW 14 kDa and 16.5 kDa) are observed in junction-enriched fiber membrane fractions. These junction-specific proteins are bound to the membrane via calcium. In addition to MIP and the 17.5 kDa protein they are possibly involved in the calcium-dependent regulation of lens fiber junctions. The 14 and 16.5 kDa proteins are also present in epithelial membranes, prepared from freshly obtained calf lens epithelia. Whereas in the latter membranes the two proteins form part of the four 14-17 kDa major protein components, these proteins are absent in membranes from cultured lens epithelial cells. The epithelial 14 kDa and 16.5 kDa proteins thus appear to be junction-specific. The capacity of the latter proteins to bind calmodulin in the presence and absence of calcium indicates that these junction-specific proteins are very similar, if not identical, to the corresponding fiber junctional proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone.

A cDNA library, prepared from poly(A+) mRNA isolated from chicken brain, was screened for calmodulin sequences using the cloned full length structural gene from Electrophorus electricus as probe (Lagacé, L., Chandra, T., Woo, S.L.C., and Means, A.R. (1983) J. Biol. Chem. 258, 1684-1688). Fifteen positive signals were detected among 4500 recombinant clones from which two overlapping clones (pCB12 and pCB15) were selected for subsequent DNA sequencing. The combined unique sequences of the two cDNA clones yielded 1395 base pairs and contained the entire coding region for calmodulin, 94 base pairs of the 5'-nontranslated region, and the entire 3'-nontranslated region of 857 base pairs. The derived amino acid sequence of chicken calmodulin is identical with that of the bovine or human protein. Compared to the eel, there is a single conservative amino acid substitution at position 74 which is occupied by Arg in the chicken and Lys in the eel. The overall nucleotide homology between the amino acid coding regions of chicken and eel calmodulin mRNA is 79%. However, the 5'- and 3'-nontranslated regions of the chicken and eel mRNA for calmodulin are highly diverged with sequence homologies of 21 and 29%, respectively. The cDNA clones were used as probes to determine the size and distribution of calmodulin mRNA in a variety of chicken tissues. In all tissues examined, two species of mRNA for calmodulin were detected at 1600 and 1900 nucleotides. Both mRNAs occurred in the cytoplasm with an abundance ratio of 4:1 for the 1600 and 1900 species, respectively. The two mRNAs appear to result from differential processing of transcripts from a single calmodulin gene. Screening of a chicken genomic phage library using pCB12 as a probe yielded a single positive designated CL-1 which contains a DNA insert of 13.5 kilobase pairs. Partial sequencing of CL-1 has confirmed the presence of sequences which code for calmodulin. A comparison of the restriction maps of CL-1 and pCB12 and pCB15 indicates that CL-1 contains at least 3 intervening sequences.

Amino Acid Sequence↗
PubMed1981

Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site.

Calmodulin is a small, acidic, calcium-binding protein that exhibits multiple in vitro biochemical activities. Although calmodulin has no known enzymatic activity, it stimulates several enzyme activities in calcium-dependent manner. Because of its ubiquitous distribution and highly conserved structure, it has been difficult to elicit anti-calmodulin sera of useful titer. We describe here a reproducible and rapid method for producing anti-calmodulin sera. This method requires the injection of performic acid-oxidized calmodulin, but the antisera react equally well with unoxidized calmodulin. A response was elicited in 11 out of 11 rabbits using three variations of this method. Antisera titers were high enough to enable development of a quantitative radioimmunoassay using dilutions of whole sera, immunoglobulin fractions, or immunoglobulin fractions purified on calmodulin-Sepharose conjugates. For the majority of the antisera, the immunoreactive site is contained in a unique region of the calmodulin molecule. Based on the quantitative reactivity of overlapping tryptic and cyanogen bromide peptides, we propose that a major immunoreactive site is fund within an 18-residue region in the COOH-terminal domain of calmodulin.

Animals↗
PubMed1981

Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation.

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1982

Interaction of beta-endorphin and other opioid peptides with calmodulin.

A highly purified preparation of calmodulin activated a calmodulin-deficient phosphodiesterase by more than 10-fold. This activation of phosphodiesterase by calmodulin was completely inhibited by two opioid peptides, beta-endorphin and dynorphin, at concentrations that had no appreciable effect on the basal phosphodiesterase activity. By contrast, similar concentrations of other structurally related peptides, including alpha-endorphin, (des-Tyr1)-gamma-endorphin, Leu-enkephalin, and Met-enkephalin, failed to block calmodulin's activation of phosphodiesterase. The inhibition by beta-endorphin of calmodulin's action was not reversed by calcium or by the opiate antagonist naloxone but was overcome by increasing the concentration of calmodulin. Equilibrium dialysis studies showed that 125I-labeled beta-endorphin bound directly to calmodulin in a saturable, calcium-dependent manner with a dissociation constant of approximately 4.6 microM. There was substantially less binding of beta-endorphin to troponin-C and little or no calcium-dependent binding of beta-endorphin to bovine serum albumin, lactalbumin, or histone. This interaction of beta-endorphin with calmodulin was similar in several respects to the interaction of certain antipsychotic drugs to calmodulin and may explain certain of the peptide's biochemical effects.

Animals↗
PubMed1984

Ophiobolin A. A natural product inhibitor of calmodulin.

Ophiobolin A, a fungal metabolite and a phytotoxin which can stimulate the net leakage of electrolytes and glucose from maize seedling roots (Tipton, C. L., Paulsen, P. V., and Betts, R. E. (1977) Plant Physiol. 59, 907-910) was found to be a potent inhibitor of calmodulin-activated cyclic nucleotide phosphodiesterase. The physiologically less active analogue, 3-anhydro-ophiobolin A, was found to be less inhibitory than ophiobolin A in the phosphodiesterase assay. The direct interaction between ophiobolin A and calmodulin has been demonstrated by changes in fluorescence of the protein and by the effect of ophiobolin A on calmodulin activity upon preincubation. Addition of ophiobolin A to calmodulin solutions resulted in an instantaneous quenching of the intrinsic tyrosine fluorescence followed by a time-dependent quenching. The instantaneous quenching is probably due to the inner filtering effect of ophiobolin A. The time-dependent fluorescence quenching was correlated with a time-dependent inhibition of calmodulin upon preincubation with ophiobolin A. The inhibition of calmodulin by ophiobolin A could not be reversed by dialysis, dilution, nor denaturation by urea in the presence of methanol followed by renaturation, and was much more pronounced in solutions containing Ca2+ than in those containing EGTA. Ophiobolin A also was shown to inhibit spinach calmodulin. The results of the present study suggest that calmodulin may be one of the target proteins of the phytotoxic action of ophiobolin A and that the interaction of ophiobolin A with calmodulin may involve a covalent modification of the protein by the fungal metabolite.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1984

The calmodulin-binding domain on microtubule-associated protein 2.

Microtubule-associated protein 2 (MAP2) binds calmodulin with a stoichiometry approaching 1-1.5 mol of calmodulin/mol of MAP2 in the presence of calcium ion. The calmodulin-binding domain(s) of MAP2 were probed by cross-linking 125I-calmodulin with partially digested MAP2, by limited digestion of the preformed 125I-calmodulin-MAP2 adduct, and by cross-linking 125I-calmodulin with the projection- and assembly-promoting portions of MAP2. Cross-linking 125I-calmodulin with partially digested MAP2 resulted in radioactive adducts of approximately 300, approximately 235, approximately 205, approximately 58, and approximately 40 kDa. The radioactive adducts with smaller molecular mass became prominent with increasing time of digestion concomitant with loss of those with higher molecular size. Limited chymotryptic digestion of preformed 125I-calmodulin-MAP2 adducts also produced a approximately 58-kDa radioactive band followed later by a approximately 40-kDa band. Brief chymotryptic digestion and subsequent centrifugation of microtubules preformed with pure tubulin and MAP2 permitted separation of microtubule-bound MAP2 fragments (molecular mass = approximately 215, approximately 180, and approximately 36 kDa) from unbound fragments (molecular mass = approximately 240, approximately 180, and approximately 140 kDa). 125I-Calmodulin cross-linked only with the microtubule-bound MAP2 fragments (forming mainly the approximately 58-kDa adduct) and not with unbound MAP2 fragments. Since the apparent molecular size of calmodulin is approximately 21 kDa on these sodium dodecyl sulfate-polyacrylamide gels, the results indicate that partial digestion of MAP2 by chymotrypsin produces a approximately 37-kDa fragment which can be further degraded to a approximately 20-kDa fragment. The approximately 37-kDa fragment that is labeled corresponds to the previously identified assembly-promoting fragment that attaches to the microtubule.

Animals↗
PubMed1983

Free energy coupling in the interactions between Ca2+, calmodulin, and phosphorylase kinase.

Interactions between Ca2+, exogenous calmodulin, and white skeletal muscle phosphorylase kinase have been quantitatively studied by equilibrium gel filtrations and analyzed by means of the so-called "linked functions" theory (Weber, G. (1975) Adv. Protein Chem. 29, 1-83). Four moles of calmodulin, each saturated with at least 3 Ca2+ ions, bind to 1 mol of phosphorylase kinase with a Kdiss of 2.3 nM. The activation of the enzyme as a function of free [Ca2+] shows that the intrinsic Ca-binding properties of phosphorylase kinase do not change upon binding of exogenous calmodulin, and confirms that alpha beta gamma delta X Ca3 is the functional catalytic unit through which activation occurs. Direct binding studies reveal that the intrinsic Ca-binding properties of the enzyme remain the same in the presence of either 0.5 or 8 mM Mg2+, indicating that phosphorylase kinase is endowed with Ca-specific sites. Upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding: whereas its first two stoichiometric Ca-binding constants are not significantly different from those of free calmodulin, the third Ca2+ ion binds with an affinity at least 10(5)-fold higher than the corresponding one in free calmodulin. Calmodulin liganded with 1 or 2 Ca2+ displays the same low affinity for the enzyme as calmodulin depleted of Ca2+ (approximate Kdiss = 10(-4)-10(-3) M). The alpha beta gamma delta X calmodulin X Ca3 complex is strengthened by a free energy coupling of -8 kcal/mol upon complexation. The quantitative analysis of our results predicts that in spite of this high free energy barrier the dissociation of the complex (i.e. the inactivation of phosphorylase kinase) occurs rapidly upon lowering free [Ca2+].

Animals↗
PubMed1982

Two opposing effects of calmodulin on microtubule assembly depend on the presence of microtubule-associated proteins.

The effect of bovine brain calmodulin on the assembly of pure bovine brain tubulin has been examined in the presence and absence of microtubule-associated proteins (MAPs). In the absence of MAPs, calmodulin enhances the rate and extent of polymerization of pure tubulin, probably by sequestering Ca2+ from tubulin since the effect is mimicked by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and parvalbumin. From stoichiometric considerations, all 4 Ca2+ binding sites of calmodulin appear to participate in this effect. In the presence of MAPs, calmodulin confers increased Ca2+ sensitivity on the tubulin polymerization process, enhancing the inhibitory effect of Ca2+ on the rate and extent of assembly. The effect of calmodulin on the assembly of tubulin is dependent on the presence of Ca2+. The data suggest that calmodulin of both low (Ca1-22+.calmodulin) and high (Ca3-42+.calmodulin) Ca2+-induced inhibition of polymerization. Thus, calmodulin has dual and opposing actions on Ca2+ sensitivity of tubulin polymerization depending on the presence or absence of MAPs.

Animals↗
PubMed1994

Calmodulin antagonistic action of new 1,5-benzothiazepines derived from diltiazem.

A series of newly synthesized 1,5-benzothiazepines derived from diltiazem (CAS 42399-41-7) were tested for calmodulin antagonistic activities using Ca(2+)-calmodulin stimulated phosphodiesterase (PDE). Some compounds possessing the benzoyloxy moieties at position 4 of 1,5-benzothiazepine ring of diltiazem showed a dose-dependent inhibitory action with the potencies comparable to that of a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). In contrast, diltiazem did not exert the inhibitory action at the same concentrations. Further, radioligand binding experiment, using a radiolabeled 1,5-benzothiazepine, showed that these compounds bound to Ca(2+)-calmodulin complex, but not to calmodulin in the presence of EGTA, suggesting that these 1,5-benzothiazepines are new calmodulin antagonists. Some of these compounds inhibited [3H]diltiazem binding to Ca antagonist binding sites in cell membranes of rat cerebral cortex but with a less potent affinities than diltiazem, suggesting that there was no correlation between their anti-calmodulin effect and the binding affinity to Ca antagonist binding sites. In conclusion, new 1,5-benzothiazepines have been demonstrated to have an anti-calmodulin action. These compounds may possess a pharmacological activity based on their anti-calmodulin action in addition to their interaction with Ca channel.

Animals↗
PubMed1993

The calmodulin-binding domain of the mouse 90-kDa heat shock protein.

The mouse 90-kDa heat shock protein (HSP90) and Ca(2+)-calmodulin were cross-linked at an equimolar ratio using a carbodiimide zero-length cross-linker. To identify the calmodulin-binding domain(s) of HSP90, CNBr-cleaved peptide fragments of HSP90 were mixed with Ca(2+)-calmodulin and cross-linked. Amino acid sequence determination revealed that an HSP90 alpha-derived peptide starting at the 486th amino acid residue was contained in the cross-linked products, which contains a calmodulin-binding motif (from Lys500 to Ile520). A similar motif is present also in HSP90 beta (from Lys491 to Val511). The synthetic peptides corresponding to these putative calmodulin-binding sequences were found to be cross-linked with Ca(2+)-calmodulin and to prevent the cross-linking of HSP90 and Ca(2+)-calmodulin. Both HSP90 alpha and HSP90 beta bind Ca2+. The HSP90 peptides bind HSP90 and thereby inhibit the binding of Ca2+. In addition, the HSP90 peptides augment the self-oligomerization of HSP90 induced at elevated temperatures. These results suggest that the calmodulin-binding domain of HSP90 might interact with another part of the same molecule and that Ca(2+)-calmodulin might modulate the structure and function of HSP90 through abolishing the intramolecular interaction.

Amino Acid Sequence↗
PubMed1998

Calcium-dependent metabolic regulations in prokaryotes indicate conserved nature of calmodulin gene.

Role of free calcium and calcium binding protein calmodulin as signal molecule in cellular regulation is well established in eukaryotes. However, reports on Ca(2+)-dependent processes and their inhibition by calcium and/or calmodulin antagonists indicate towards the presence of calmodulin in prokaryotes as well. The common evolutionary origin of pro- and eukaryotes and many examples of evolutionary conservation of structure and functions support the contention of such conservation of the role of Ca2+ and calmodulin. Eukaryotic calmodulin (CaM) contains four structurally and functionally similar Ca2+ domains named I, II, III and IV. Each Ca2+ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of Ca2+ binding. Plant calmodulin differ from vertebrate ones in 13 to 14 amino acid positions of which nine occur at -COOH- terminal half. Differences between protozoan and mammalian CaM also occur mostly in the same half. Isolation and characterization, although to a little extent, of CaM-like proteins from bacteria and cyanobacteria and their comparison with CaMs from diverse origin suggest high degree of conservation. Non-bulky amino acids like glycine, alanine and serine with low specific rotation are present in greater number in the primitive form of calmodulin and have been significantly reduced in highly evolved form of calmodulin, suggesting that their requirement was insignificant and were eliminated from EF hand structure during evolution. However, amino acids like glutamate/glutamine and aspartate/asparagine were highly conserved and did not show any major change in their frequency since their positions are too significant in calcium binding domain. While the number of positively charged amino acids like arginine and leucine was increased, histidine containing weakly ionized group and having a significant buffering capacity was reduced to a major extent, further suggesting that the acidic nature of calmodulin protein has been maintained during evolution. Thus it is now clear that the entire superfamily of Ca2+ binding proteins have arisen from a common genetic ancestry. Two successive tandem duplications of gene encoding a single domain containing protein of 30-40 residues gave rise to a four domain molecule from which this family was then derived.

Amino Acid Sequence↗
PubMed1995

Interaction of smooth muscle caldesmon with calmodulin mutants.

The interaction of avian smooth muscle caldesmon with calmodulin (CaM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to caldesmon was decreased 2-4 times by point mutation G33V-CaM, double mutation E84K/E120K-CaM, deletion of residues 82-84, and by cluster mutations DEE118-120-->KKK or EEE82-84-->KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84-->KKK and DEE118-120-->KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to caldesmon, and that Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon.

Animals↗
PubMed1990

Calmodulin-binding proteins and calcium/calmodulin-regulated enzyme activities associated with brain actomyosin.

Calcium- and calmodulin-regulated ATPase and protein kinase activities are shown to be strongly associated with brain actomyosin. Similar enzymatic activities and an invariable polypeptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were obtained for brain actomyosin taken through a solubilization-precipitation cycle (1.0-0.1 M KCl), or precipitated from buffers containing 1% Triton X-100 or 10 mM EDTA and 10 mM EGTA. These data suggest a specific complex of brain actomyosin with a protein kinase similar to calmodulin-dependent kinase II, a 190-kDa calmodulin-binding protein (P190), and a calmodulin-like polypeptide. P190 was the major substrate for endogenous calcium-dependent phosphorylation. 125I-Calmodulin overlay technique revealed four major calmodulin-binding polypeptides associated with brain actomyosin: 50- and 60-kDa subunits of the calmodulin-dependent kinase II, P190, and a high molecular weight polypeptide which is probably fodrin. A fraction enriched in P190 had Ca2(+)- and calmodulin-stimulated MgATPase activity, but not myosin-like K-EDTA ATPase activity. The lack of immunological cross-reactivity between brain myosin heavy chain and P190 confirmed that they are distinct molecules.

Actomyosin↗