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M Comte

Publications and source records attributed to M Comte.

At least 19 recordsLinked to original sources
PubMed1995

Thermodynamic and molecular properties of the interaction between amphioxus calcium vector protein and its 26 kDa target.

Calcium vector protein (CaVP) of amphioxus shares some common structural features with Ca(2+)-dependent activators such as troponin C and calmodulin, and is associated in vivo with a 26 kDa (CaVPT), a multidomain protein with one IQ- and two IgII-motifs. Isolated CaVP binds two Ca2+ ions with very different intrinsic affinity constants: K'Ca1 = 4.9 x 10(6) M-1 and K'Ca2 = 7.3 x 10(3) M-1, respectively. In the complex with CaVPT, CaVP also binds two Ca2+, but with strong positive cooperativity (nH = 1.9) and with distinctly higher affinity: K'Ca1 = 2.4 x 10(5) M-1 and K'Ca2 = 1.0 x 10(8) M-1. Since neither in the isolated CaVP nor in the complex Ca2+ binding is influenced by 2 mM MgCl2, both sites can be considered as Ca(2+)-specific. In the absence of Ca2+, the complex is stable under physiological conditions, but the interaction is governed by the principle of linked functions and Ca2+ binding to CaVP reinforces the affinity between CaVP and CaVPT 70-fold. Both proteins interact with the hydrophobic probe 2 p-toluidinylnaphthalene-6-sulfonate (TNS), but CaVPT enhances the fluorescence 45-fold, CaVP-Ca2 and metal-free CaVP only 10- and 5-fold, respectively. Complex formation between CaVPT and CaVP leads to a 3-fold reduction of the fluorescence enhancement, suggesting that a strong solvent-shielded hydrophobic core is formed. CaVP contains two highly reactional thiols (kSH > 0.3 s-1) for 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); CaVPT contains three thiols, two of them also with kSH > 0.3 s-1 in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina.

Neural Ca(2+)-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin delta, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner retina, i.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.

Animals↗
PubMed1994

Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins.

VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Characterization and primary structure of amphioxus troponin C.

Troponin C (TnC) from amphioxus (Protochordate) was purified and its primary structure determined. Unlike the case of vertebrates and other invertebrates, amphioxus TnC is found in the soluble fraction after extractions at physiological ionic strength in the presence of Ca2+. Edman sequencing combined with mass spectroscopy indicate that the protein contains 163 amino acid residues. It possesses an acetylated N-terminus (although a small percentage has a free Ser N-terminus) and either epsilon-N-methyllysine or epsilon-N-dimethyllysine in position 20. It displays about 50% sequence identity with vertebrate skeletal-muscle and cardiac-muscle TnC, 44% with TnC of sea squirt, also a Protochordate, and 30% with other invertebrate TnC. Like vertebrate TnC, amphioxus TnC contains a N-terminal alpha-helix plus the usual four ancestral Ca(2+)-binding regions, but analysis of the sequence suggests that the fourth site is not functional. Flow dialysis shows that amphioxus TnC binds three Ca2+ with the mean apparent affinity constant K' of 3.4 +/- 1.5 10(5) M-1. No cooperativity exists between the sites, and the presence of up to 10 mM Mg2+ does not influence the Ca(2+)-binding isotherm, indicating that the metal-binding sites are Ca(2+)-specific at physiological Mg2+ concentrations. It forms a Ca(2+)-dependent, 1:1 complex with melittin and rabbit or crayfish troponin I (TnI). Amphioxus TnC possesses one Trp residue in position 151 and one at the C-terminus. Trp fluorescence suggests that one or both residues are solvent-exposed in the metal-free form and efficiently shielded in the Ca2+ form. Although Mg2+ has no effect on the Ca2+ binding, the Trp fluorescence is influenced by millimolar Mg2+, suggesting the presence of one or more independent Mg(2+)-binding site(s). A phylogenetic analysis clearly shows that amphioxus TnC is positioned on the branch of the Chordates, but at a distance from the vertebrate TnC. Its place on the phylogenetic tree is in accordance with the consensus evolutionary phylogeny.

Amino Acid Sequence↗
PubMed1993

Immunolocalization of calcium vector protein and its target protein in amphioxus.

Three proteins, sarcoplasmic Ca(2+)-binding protein (SCP), Ca2+ vector protein (CaVP) and its target protein (CaVPT), are found abundantly in the higher invertebrate amphioxus. Whereas the function of SCP is likely to be related to Ca2+ and Mg2+ buffering, that of the latter two proteins, apparently linked together, is still not clear. In this study, affinity-purified polyclonal antibodies to these three proteins were used to study the extractability under physiological ionic conditions, the distribution in different tissues and the immunocytochemical localization in striated muscle. Our data show that SCP is essentially cytosolic whereas CaVP and CaVPT are partially associated with non-soluble components in amphioxus tissues. The tissue distribution, studied in transverse sections, shows that SCP is merely confined to striated muscle, whereas CaVP and CaVPT are also abundant in other tissues such as the spinal chord and the gonads. Thus the protein pair CaVP/CaVPT is likely to serve a general role in many tissues; however, no strict correlation was found in the distribution of the latter two proteins, suggesting that they may function independently. The detailed cytochemical localization of the three proteins in longitudinal sections of striated muscle revealed a discrete striation pattern in addition to a diffuse background. For SCP these striations are coincident with the Z line. The immunostaining for CaVP shows intense striations at the level of the Z lines alternating with weak striations at the M lines. For CaVPT the striations at the Z and M line are more or less of equal intensity, leading to a pattern with a 1 micron periodicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.

Animals↗
PubMed1989

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry.

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.

Animals↗
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↗
PubMed1987

High-affinity formation of a 2:1 complex between gramicidin S and calmodulin.

Two molecules of gramicidin S, a very rigid cyclic decapeptide rich in beta-sheet structure, can bind in a Ca2+-dependent way to a calmodulin molecule in the presence as well as in the absence of 4 M-urea. The flow-microcalorimetric titration of 25 microM-calmodulin with gramicidin S at 25 degrees C is endothermic for 21.3 kJ.mol-1; the enthalpy change is strictly linear up to a ratio of 2, indicating that the affinity constant for binding of the second gramicidin S is at least 10(7) M-1. In 4 M-urea the peptide quantitatively displaces seminalplasmin from calmodulin, as monitored by tryptophan fluorescence. An iterative data treatment of these competition experiments revealed strong positive co-operativity with K1 less than 5 X 10(5) M-1 and K1.K2 = 2.8 X 10(12) M-2. A competition assay with the use of immobilized melittin enabled us to monitor separately the binding of the second gramicidin S molecule: the K2 value is 1.9 X 10(7) M-1. By complementarity, the K1 value is 1.5 X 10(5) M-1. In the absence of urea the seminalplasmin displacement is incomplete: the data analysis shows optimal fitting with K1 less than 2 X 10(4) M-1 and K1.K2 = 3.2 X 10(11) M-2 and reveals that the mixed complex (calmodulin-seminalplasmin-gramicidin S) is quite stable and is even not fully displaced from calmodulin at high concentrations of gramicidin S. The activation of bovine brain phosphodiesterase by calmodulin is not impaired up to 0.2 microM-gramicidin S. According to our model the ternary complex enzyme-calmodulin-gramicidin is relatively important and displays the same activity as the binary complex enzyme-calmodulin. Gramicidin S also displaces melittin from calmodulin synergistically, as monitored by c.d. Our studies with gramicidin S reveal the importance of multipoint attachments in interactions involving calmodulin and confirm the heterotropic co-operativity in the binding of calmodulin antagonists first demonstrated by Johnson [(1983) Biochem. Biophys. Res. Commun. 112, 787-793].

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1987

Calcium-calmodulin stimulates inositol 1,4,5-trisphosphate kinase activity from insulin-secreting RINm5F cells.

In a cytosolic fraction derived from insulin-secreting RINm5F cells, the rate of conversion of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) was half-maximally stimulated by 0.8 microM Ca2+ (Biden, T. J., and Wollheim, C. B. (1986) J. Biol. Chem. 261, 11931-11934). In the present study we show that after initial purification by anion exchange chromatography, the Ins-1,4,5-P3 kinase activity responsible for that conversion is stimulated by Ca2+-calmodulin, but not by Ca2+ alone. This is almost certainly due to a specific interaction of the enzyme and its activator since kinase activity was retained on a calmodulin-linked Sepharose 6B column in the presence of Ca2+ but eluted upon chelation of the cation. After this two-step purification, Ins-1,4,5-P3 kinase activity was maximally stimulated 5-fold by 10 microM calmodulin in the presence of 10(-5) M Ca2+, and 2 1/2-fold at 10(-6) M Ca2+. Under these conditions the minimum concentrations of calmodulin needed to stimulate activity were in the 10-50 nM range. At 10(-7) M Ca2+, calmodulin (up to 30 microM) was without effect. Stimulated Ins-1,4,5-P3 kinase activity was inhibited in a dose-dependent fashion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) although the calmodulin antagonist had no effect on the residual activity seen at 10(-7) M Ca2+. These results strongly support our previous suggestion that alterations in cytosolic free Ca2+ concentrations play an important role in regulating the levels of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 during cellular stimulation.

Calcium↗
PubMed1987

Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin.

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.

Animals↗
PubMed1986

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.

Adenylyl Cyclases↗
PubMed1986

Calcium-proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses.

Microcalorimetry, pH potentiometry, and direct binding studies by equilibrium dialysis or gel filtration were performed to determine the thermodynamic functions delta Ho, delta Go, and delta So guiding the interactions of Ca2+, Mg2+, and H+ with bovine brain calmodulin. At pH 7.5, Ca2+ and Mg2+ binding are both endothermic with enthalpy changes of 19.5 and 72.8 kJ X (mol of calmodulin)-1, respectively. These enthalpy changes are identical for each of the four ion-binding domains. The affinity constants also are identical with intrinsic values of 10(5) M-1 for Ca2+ and 140 M-1 for Mg2+. Ca2+ and Mg2+ do not compete for the same binding sites: at high concentrations of both ions, a calmodulin-Ca4-Mg4 species is formed with an enthalpy value of 24.4 kJ X mol-1 with respect to calmodulin-Ca4 and -28.8 kJ X mol-1 with respect to calmodulin-Mg4. Moreover, in the presence of high concentrations of Ca2+, the affinity of each of the four ion-binding domains in calmodulin for Mg2+ is decreased by a factor of 4 and vice versa, indicative of negative free-energy coupling between Ca2+ and Mg2+ binding. Protons antagonize Ca2+ and Mg2+ binding in a different manner. Ca2+-H+ antagonism is identical in each of the four Ca2+-binding domains in the pH range 7.5-5.2. Our analyses suggest that three chemical geometries, probably carboxyl-carboxylate interactions, are responsible for this antagonism with ionization constants of 10(6.2) M-1 in the metal-free protein. Mg2+-H+ antagonism also is identical for each of the Mg2+-binding sites but is qualitatively different from Ca2+-H+ antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

The interaction of calmodulin with amphiphilic peptides.

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.

Amino Acids↗
PubMed1983

Ca2+-dependent high-affinity complex formation between calmodulin and melittin.

The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.

Animals↗
PubMed1982

Activation of human erythrocyte Ca2+-dependent Mg2+-activated ATPase by calmodulin and calcium: quantitative analysis.

The effect of Ca2+ and calmodulin on (CaM) on the activation of Ca2+-dependent Mg2+-activated ATPase (Ca2+,Mg2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) has been carried out because of the finding that the CaM dependence of the activation varies with the concentration of free Ca2+, similarly to brain phosphodiesterase and adenylate cyclase. The study was carried out in the absence of chelating agents because they strongly interfere in the enzyme kinetics. Three main conclusions can be drawn (i) CaM-Ca3 and CaM-Ca4 together are the biochemically active species in vitro. (ii) These species bind in a non-cooperative way to the CaM-binding site of the enzyme with a dissociation constant of 6 x 10(-10) M or 1.1 x 10(-8) M, depending on whether Ca2+ saturates the substrate binding site of the enzyme or not. (iii) The binding of CaM-Ca3 to the enzyme lowers the dissociation constant of the enzyme for Ca2+ at the substrate binding site from 51.5 to 2.8 microM. Contrary to general belief, CaM does not induce pronounced positive cooperativity in the binding of Ca2+ to the enzyme. Such a cooperativity is seen only when the enzyme is incompletely saturated with the activator, but it disappears in the presence of saturating concentrations of CaM-Ca3. The rate equation proposed here accurately predicts the extent of enzyme activation over a wide range of Ca2+ and CaM concentration. In healthy erythrocytes the concentrations of Ca2+ and CaM are such that the Ca pump works with a minimal dissipation of energy, but a small increase in the intracellular Ca2+ concentration leads to a strong amplification of the pumping activity.

Binding Sites↗