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PubMed1987

Calmodulin and calmodulin-binding proteins in the renal brush border.

The calmodulin content of renal brush-border membrane vesicles, prepared by Mg2+-precipitation in EGTA-containing solutions, amounts to 1.8 micrograms per mg protein. The amount and the distribution of this EGTA-insensitive calmodulin was determined in membrane and cytoskeletal fractions prepared from the brush-border membrane vesicles by extraction with Triton X-100. The Triton X-100 insoluble pellet contains 21.2% of the protein and 52.2% of the EGTA-insensitive calmodulin, which amounts in this fraction to 4.4 micrograms per mg protein. Treatment of the Triton X-100 insoluble pellet, consisting of the microvillar core residue, with ATP and Mg2+ results in the solubilization of a relatively small number of proteins among which are actin, myosin, calmodulin and several calmodulin-binding proteins. The solubilization is partially reversible and a fraction of the proteins can be precipitated by centrifugation after the enzymatic hydrolysis of ATP. Readdition of ATP to the pellet results in the resolubilization of myosin, part of the actin, an 115-kDa calmodulin-binding protein and calmodulin. The calmodulin content of the final extract was 61.8 micrograms per mg protein. We have found roughly the same distribution pattern of calmodulin and ATP-solubilized, calmodulin-binding proteins in renal and intestinal brush-border preparations. The calmodulin content, however, as well as the relative amount of the calmodulin-binding proteins versus actin are about 4 to 5-times higher in intestinal than in renal microvillar core residues.

Adenosine Triphosphate↗
PubMed1985

Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B.

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.

Animals↗
PubMed1992

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.

Amino Acid Sequence↗
PubMed1996

Evidence for a role of calmodulin in calcium-induced noradrenaline release from permeated synaptosomes: effects of calmodulin antibodies and antagonists.

The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca(2+)-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca2+/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca(2+)-induced nor-adrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca(2+)-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca(2+)-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca(2+)-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca2+/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.

Animals↗
PubMed1987

Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin.

Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin. The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol). The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added. On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin. Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.

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

Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase.

Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.

Adenosine Triphosphate↗
PubMed1988

Functional in vitro test of calmodulin antagonism: effect of drugs on interaction between calmodulin and glycolytic enzymes.

A simple procedure has been elaborated to screen for the calmodulin antagonist effect of drugs. A covalently attached fluorescent probe was used to monitor the binding of enzymes known as target enzymes to calmodulin. Moreover, the probe made it possible to recognize a new target enzyme, aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), for calmodulin among glycolytic enzymes. The calmodulin antagonist trifluoperazine prevented or eliminated the complex formation between calmodulin and enzymes studied in reconstituted systems; the Ca channel blockers had no effect. The functional consequences of the effect of drugs on calmodulin-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) interaction were investigated as well. Whereas trifluoperazine suspended the calmodulin-mediated hysteretic inactivation of phosphofructokinase, Ca channel blockers (verapamil and nifedipine) were ineffective. Fendiline (regarded as a Ca channel blocker) seems to act as a functional calmodulin antagonist. Its binding to calmodulin does not prevent the complex formation of phosphofructokinase and calmodulin, but within this ternary complex phosphofructokinase preserves or recovers its original activity measured in the absence of calmodulin. The possible molecular effect of drugs on a calmodulin-enzyme complex is discussed.

Calcium Channel Blockers↗
PubMed1987

Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin.

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.

Amino Acid Sequence↗
PubMed1988

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity.

The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the 110K-calmodulin complex with alpha-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actin-binding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrow-head appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium- or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at approximately 40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with alpha-chymotrypsin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATP. Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the 110-kD polypeptide. The 78 kD, two of the three fragments at approximately 40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the 110K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.

Actins↗
PubMed1992

The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid.

Peptide G25 (KKAVKVPKKEKSVLQGKLTRLAVQI) representing the putative lipid-binding region (G region) of the erythrocyte Ca2+ pump was synthesized. This peptide interacted with acidic lipids, as shown by the increase in size of phosphatidylserine liposomes in its presence. This lipid interaction is consistent with the previous evidence suggesting that the portion of the pump from which this peptide was taken is responsible for the activation of the pump by acidic lipid. G25 also bound to calmodulin, as was shown by its cause of a shift in the fluorescence of 5-dimethylamino naphthalene-1-sulfonyl- (dansyl)-calmodulin, and by its competition with Ca2+ pump for calmodulin. Its Kd for dansyl-calmodulin was much higher (0.8 microM) than that of the peptides representing the primary calmodulin-binding region (C region) of the Ca2+ pump. Although the presence of the G region provided the possibility of a second calmodulin-binding site, activation of the pump by calmodulin always could be fitted by simple saturation kinetics. The calmodulin-binding peptide from the C region of the pump, C28R2, also interacted with lipid with even greater effectiveness than G25. When the C region of the pump was saturated with calmodulin, acidic lipid activation of the pump followed simple saturation kinetics. However, when calmodulin was omitted, a higher concentration of lipid was needed for saturation and the kinetics became complex. The data are consistent with the idea that calmodulin activates the pump only by interaction at the C region, but that acidic lipid activates by interaction at both of the C and G regions.

Animals↗
PubMed1984

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide).

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.

Animals↗
PubMed1982

Trifluoperazine inhibition of calmodulin-sensitive Ca2+ -ATPase and calmodulin insensitive (Na+ +K+)- and Mg2+ -ATPase activities of human and rat red blood cells.

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+ -ATPase and calmodulin-insensitive (Na+ +K+)- and Mg2+ -ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+ -ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+ -ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ +K+)-ATPase and Mg2+ -ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.

Adenosine Triphosphatases↗
PubMed1983

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum.

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.

Animals↗
PubMed1985

Interaction of calmodulin and a calmodulin-binding peptide from myosin light chain kinase: major spectral changes in both occur as the result of complex formation.

Many different enzymes are activated by direct interaction with calmodulin; this interaction is thought to occur through a distinct calmodulin-binding domain in each of these enzymes. We have recently reported the sequence of a 27-residue peptide (denoted M13), derived from skeletal muscle myosin light chain kinase (MLCK), that exhibits the properties expected of a calmodulin-binding domain [Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191]. The interaction between chemically synthesized M13 peptide and calmodulin has been studied by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) spectroscopy. In the presence of Ca2+, the observed ellipticity of an equimolar mixture of M13 and calmodulin is much greater than the sum of the ellipticities of the two isolated proteins. In the absence of Ca2+, the measured ellipticity of the mixture is approximately the sum of the two components. Addition of the peptide to calmodulin causes dramatic changes in the proton NMR spectrum; at a 1:1 molar ratio, no evidence of either free peptide or free calmodulin is observed. Moreover, these data demonstrate that a unique species of the M13-calmodulin complex is formed, indicating that the peptide binds to calmodulin in only one way. The many resonances affected by M13 binding include residues in both halves of the calmodulin molecule. The observed CD and NMR effects suggest that secondary and tertiary conformational changes occur both in M13 and in calmodulin upon complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase.

A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase.

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.

Animals↗
PubMed1997

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% +/- 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 +/- 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Animals↗
PubMed1983

A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells.

We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.

Animals↗