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PubMed2001

Detection of calmodulin-binding proteins and calmodulin-dependent phosphorylation linked to calmodulin-dependent chemotaxis to folic and cAMP in Dictyostelium.

Calmodulin (CaM) antagonists, trifluoperazine (TFP) or calmidazolium (R24571), dose-dependently inhibited cAMP and folic acid (FA) chemotaxis in Dictyostelium. Developing, starved, and refed cells were compared to determine if certain CaM-binding proteins (CaMBPs) and CaM-dependent phosphorylation events could be identified as potential downstream effectors. Recombinant CaM ([35S]VU-1-CaM) gel overlays coupled with cell fractionation revealed at least three dozen Ca(2+)-dependent and around 12 Ca(2+)-independent CaMBPs in Dictyostelium. The CaMBPs associated with early development were also found in experimentally starved cells (cAMP chemotaxis), but were different for the CaMBP population linked to growth-phase cells (FA chemotaxis). Probing Western blots with phosphoserine antibodies revealed several phosphoprotein bands that displayed increases when cAMP-responsive cells were treated with TFP. In FA-responsive cells, several but distinct phosphoproteins decreased when treated with TFP. These data show that unique CaMBPs are present in growing, FA-chemosensitive cells vs. starved cAMP-chemoresponsive cells that may be important for mediating CaM-dependent events during chemotaxis.

Animals↗
PubMed1984

Calmodulin in human serum and the specific release of calmodulin from calmodulin-rich platelets.

Calmodulin (CaM)-like activity was detected in human serum and foetal calf serum, with an activity 10 times more than that detectable in plasma. Serum CaM was largely accounted for by release from human platelets as confirmed by both radioimmunoassay and sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis of CaM partially purified from platelet releasate obtained in response to thrombin. Lactate dehydrogenase release was unaffected by thrombin. Platelet CaM content was very variable (1.3 to 11.3 micrograms/mg protein; n = 15). It is suggested that intact platelets are rich in CaM and that release of CaM during preparation explains the variation in CaM content reported here and in the literature.

Adult↗
PubMed1999

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.

Binding, Competitive↗
PubMed2001

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein.

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.

Animals↗
PubMed1986

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody.

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.

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

Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells.

A calmodulin-like protein, which is identical in size and 85% identical to vertebrate calmodulin, was recently identified by 'subtractive hybridization' comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of calmodulin, calmodulin-like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, calmodulin-like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare calmodulin-like protein functions with those of calmodulin. We find that calmodulin-like protein activation of multifunctional Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II) is equivalent to activation by calmodulin, but that four other calmodulin-dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric-oxide synthase, and myosin-light-chain kinase, display much weaker activation by calmodulin-like protein than by calmodulin. In the case of myosin-light-chain kinase, calmodulin-like protein competitively inhibits calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, calmodulin-like protein may have evolved to function as a specific agonist of certain calmodulin-dependent enzymes, and/or as a specific competitive antagonist of other calmodulin-dependent enzymes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1984

Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins.

The molecular mechanisms by which calcium regulates cellular processes such as metabolic and mechanochemical events probably involve interactions with a variety of molecules. A large body of evidence suggests that the targets of calcium's regulatory effects inside the cell are calcium-binding proteins. Our work attempts to correlate calcium-binding protein structure with activities. In this chapter we have presented some of our recent studies on these functional domains in calmodulin and calmodulin-binding proteins. Selected chemical modifications of known amino acid sequence positions have demonstrated the presence of multiple functional domains on calmodulin, have allowed the dissociation of calmodulin functions, and have provided the necessary tools for further investigations of the molecular basis of calmodulin action. One of these modifications, iodination of tyrosine-99, has allowed us to develop procedures to reproducibly detect calmodulin-binding proteins by a binding technique. This method is technically simple. It allows us to detect and study calmodulin-binding proteins (e.g., myosin heavy chain and membrane gap junction proteins) that would be difficult to study with immobilized calmodulin. We have developed a library of antibodies to calmodulin and related proteins such as troponin C and S100 beta. Some of these antisera appear to be site-specific gamma globulins. We have demonstrated that reactivity can be contained in an amino acid sequence as short as seven residues. We have demonstrated the feasibility of using an immunochemical mapping approach to study calmodulin and calmodulin-binding proteins. Comparative sequence analyses combined with functional analyses have allowed correlation of function and structure and have suggested logical candidates for functional domains on calmodulin and calmodulin-binding proteins. Although not discussed in detail in this chapter, these calmodulin-binding proteins appear to contain amino acid sequence homologies. This suggests, analogous to the approach used for calmodulin and related proteins, a logical starting point for domain analyses of calmodulin-binding proteins. Interestingly, structural homologies among calmodulin-binding proteins are reminiscent of the different phosphorylation sites found in many of the physiological substrates for protein kinases. Since a number of calmodulin-binding proteins are themselves substrates for protein kinases, these results suggest another possible point of interrelationships between calcium and cyclic nucleotide regulation.

Amino Acid Sequence↗
PubMed1994

Repeated haloperidol increases both calmodulin and a calmodulin-binding protein in rat striatum.

Repeated treatment with the antipsychotic drug, haloperidol, leads to an increased behavioral sensitivity to dopamine agonists exhibited upon withdrawal from the drug. An increase in the particulate content of the endogenous Ca(2+)-binding protein, calmodulin, has been demonstrated after repeated treatment of rats with haloperidol. In this study, the anatomical specificity of the effect of repeated haloperidol treatment on the content and subcellular localization of calmodulin was investigated. Responsivity of calmodulin localization to dopaminergic input following drug treatment was assessed by determining the subcellular localization of calmodulin following an in vivo amphetamine challenge before sacrifice. Male, Sprague-Dawley rats were treated with 0.5 mg/kg haloperidol (s.c.) for 3 weeks and withdrawn from the drug for 4 days. Repeated haloperidol increased calmodulin content only in the striatum but altered the subcellular distribution of calmodulin in rat limbic forebrain and frontal cortex. In the latter areas, the soluble calmodulin was increased while the particulate calmodulin was decreased. There was no change in calmodulin in either hippocampus or cerebellum in response to drug treatment. Challenge with the dopamine mimetic, amphetamine, before sacrifice was effective in redistributing calmodulin only in striatum from rats that had been treated repeatedly with haloperidol, demonstrating an increased sensitivity of the translocation process. In order to determine whether a change in a calmodulin-binding protein would accompany the drug-induced increase in calmodulin, striatal calmodulin-binding proteins were examined using a biotinylated calmodulin overlay technique. Repeated haloperidol treatment enhanced calmodulin binding to a 150 kDa protein in striatal membranes. The 150 kDa protein exhibited the same gel mobility and subcellular distribution as myosin light chain kinase immunoreactivity. There was an increase in myosin light chain kinase immunoreactivity in striatal membranes after repeated haloperidol that was apparent in animals withdrawn either 4 or 10 days from haloperidol treatment. Therefore, repeated haloperidol could increase the rat striatal content of calmodulin and potentially that of the calmodulin-binding protein, myosin light chain kinase. Increases in striatal calmodulin and myosin light chain kinase may signal a greatly enhanced sensitivity of actin-myosin interactions after repeated haloperidol that could contribute to haloperidol-induced neurochemical or morphological changes involved in drug-induced synaptic plasticity.

Amphetamine↗
PubMed1996

Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth.

The role calmodulin plays in the growth and differentiation of nerve cells was assessed by altering the levels of calmodulin in the PC12 rat pheochromocytoma cell line and determining the effects of altering these levels on cellular proliferation and differentiation. Calmodulin levels in the PC12 cells were increased or decreased by transfecting the cells with a mammalian expression vector into which the rat calmodulin gene I had been cloned in the sense or antisense orientation, respectively. The cells transfected with the calmodulin sense gene showed increased levels of calmodulin immunoreactivity and increased levels of calmodulin messenger RNA as ascertained by immunocytochemistry and slot-blot analysis, respectively. Cells transfected with the calmodulin antisense construct showed reduced levels of calmodulin immunoreactivity. Reducing the levels of calmodulin by expression of antisense calmodulin messenger RNA resulted in a marked inhibition of cell growth, whereas increasing the levels of calmodulin by overexpressing calmodulin messenger RNA resulted in an acceleration of cell growth. Transfected PC12 cells having reduced levels of calmodulin immunoreactivity exhibited spontaneous outgrowth of long, stable and highly branched neuritic processes. PC12 cells in which calmodulin was overexpressed showed no apparent changes in cell morphology, but did show an altered response to the addition of nerve growth factor. While nerve growth factor slowed cellular proliferation and induced extensive neurite outgrowth, in parental PC12 cells nerve growth factor induced little or no neurite outgrowth and little inhibition of cell proliferation in transfected cells overexpressing calmodulin. These results indicate that calmodulin is essential for the proliferation of nerve cells and for the morphological changes that nerve cells undergo during differentiation. The study also suggests the possibility that a calmodulin antisense approach may be used to inhibit the proliferation of neuronal tumors.

Animals↗
PubMed1980

Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins.

Calmodulin is the name proposed for a multifunctional, calcium binding protein whose presence has been detected in a number of eukaryotic cells. In the studies summarized here, calmodulin has been isolated from spinach leaves (Spinacea oleracea), characterized, and compared to vertebrate calmodulins. Quantitative recovery data for a rapid-isolation protocol demonstrate that calmodulin is a major constituent of spinach leaves. Spinach calmodulin is indistinguishable from vertebrate calmodulins in phosphodiesterase activator activity using vertebrate brain phosphodiesterase and in quantitative immunoreactivity using antiserum made against vertebrate calmodulin. However, spinach calmodulin is really distinguished from vertebrate and invertebrate calmodulins in electrophoretic mobility and in amino acid composition. Spinach calmodulin, like vertebrate calmodulins, lacks tryptophan and contains 1 mol each of N epsilon-trimethyllysine and histidine per 17000 g of protein. In contrast to vertebrate calmodulins, spinach calmodulin has only one tyrosinyl residue and has a threonine/serine ratio of 1.3. While amino acid compositions indicate differences between spinach and vertebrate calmodulins, isolation and characterization of tryptic peptides containing the single histidinyl and N epsilon-trimethyllysyl residues and both prolinyl residues indicate that these regions in spinach calmodulin are similar to the corresponding regions in vertebrate calmodulin. These studies more fully define the general and specific characteristics of calmodulins and indicate that calmodulin structure is not as highly conserved among all eukaryotes as it is among vertebrates and invertebrates.

Amino Acids↗
PubMed1990

Multiple ubiquitination of calmodulin results in one polyubiquitin chain linked to calmodulin.

In the presence of Ca2+ and ATP/Mg2+ mammalian calmodulin can be covalently coupled to ubiquitin by ubiquityl calmodulin synthetase (uCaM-synthetase). Three ubiquitin derivatives 125I-CT-ubiquitin (prepared by the chloramine-T method), 125I-BH-ubiquitin (prepared by the Bolton-Hunter method) and methylated forms of ubiquitin were tested with native calmodulin. Alternatively native ubiquitin was tested with the Bolton-Hunter derivative of calmodulin (125I-BH-calmodulin). Up to three molecules of ubiquitin can be incorporated into one molecule of calmodulin. Since both native forms of ubiquitin and calmodulin are good substrates of uCaM-synthetase, ubiquitination is not a result of an altered conformation (i.e. denaturation) of either protein. With 125I-BH-calmodulin it is demonstrated that calmodulin is also present in the higher molecular weight ubiquitin conjugates. If methylated ubiquitin is employed as substrate for uCaM-synthetase only one conjugate corresponding to the mono-ubiquitination product of calmodulin is formed. This demonstrates that only a single lysine residue in calmodulin is conjugated to ubiquitin. All other higher molecular weight ubiquitin-calmodulin conjugates must therefore be composed of one molecule of calmodulin to which an oligo- or poly-ubiquitin chain is linked. Since it can be shown that the mono-ubiquitination product of calmodulin still contains ca. 1 mol trimethyllysine/mol calmodulin, the poly-ubiquitin chain is not linked to lysine 115 of calmodulin. In addition a demethylation of trimethyllysine 115 by enzymes in reticulocyte lysate or the DEAE-enriched enzyme fraction with subsequent ubiquitination at this site of calmodulin can also be excluded.

Amino Acids↗
PubMed1987

Differential inhibition of calmodulin-sensitive phosphodiesterase and Ca++-adenosine triphosphatase by chlorpromazine-linked calmodulin.

Upon irradiation with UV light, chlorpromazine binds irreversibly to calmodulin and inactivates it. To determine whether this chlorpromazine-calmodulin (CPZ-CaM) complex can inhibit the actions of native calmodulin, we examined its effects on the activity of calmodulin-sensitive cyclic nucleotide phosphodiesterase from rat brain and on the Ca++-adenosine triphosphatase (ATPase) of human erythrocyte membranes. The CPZ-CaM complex was prepared by irradiating purified bovine brain calmodulin in the presence of chlorpromazine and Ca++. The sample was then dialyzed extensively to remove reversibly bound chlorpromazine and then assayed for its ability to activate calmodulin-sensitive phosphodiesterase and Ca++-ATPase, and for its ability to block the stimulatory effects of native calmodulin on these enzymes. The CPZ-CaM complex had no effect on the basal activity of either enzyme; it neither activated nor inhibited the enzymes when assayed in the absence of calmodulin. However, it affected differentially the activation of the two enzymes by native calmodulin. The CPZ-CaM complex totally inhibited calmodulin-stimulated phosphodiesterase but had no effect on the activation of the ATPase by calmodulin. Other studies showed that CPZ-CaM increased the activation constant (Ka) for the interaction of calmodulin with phosphodiesterase but did not affect the maximal activation (Vmax) of the enzyme by calmodulin. Neither calmodulin nor CPZ-CaM altered the Km for the interaction between phosphodiesterase and cyclic AMP. These results suggest that CPZ-CaM inhibits the calmodulin-induced activation of phosphodiesterase by competing with calmodulin for regulatory sites on the enzyme and not by interacting with calmodulin itself or by blocking the interaction of cyclic AMP with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1.

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.

Amino Acid Sequence↗
PubMed1985

Calcium-dependent calmodulin binding to chromaffin granule membranes: presence of a 65-kilodalton calmodulin-binding protein.

The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.

Adrenal Medulla↗
PubMed1991

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies.

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.

Animals↗
PubMed2000

Differential regulation of calmodulin content and calmodulin messenger RNA levels by acute and repeated, intermittent amphetamine in dopaminergic terminal and midbrain areas.

Repeated doses of psychoactive drugs often produce adaptive responses that differ from the initial drug application and additional adaptive processes occur following cessation of the drug. The relationship between alterations in calmodulin protein and messenger RNA produced by an initial versus a repeated dose of amphetamine was examined, as well as changes following drug cessation. Calmodulin protein and messenger RNA of the three individual calmodulin genes were measured in rat dopaminergic cell body and terminal areas following acute or repeated amphetamine. Rats were either injected once with 2.5mg/kg amphetamine or saline and decapitated after 3h, or given 10 injections of amphetamine three to four days apart and decapitated 3h after the final injection. Calmodulin messenger RNA and protein were also measured three and seven days after ceasing drug treatment. Acute amphetamine increased calmodulin 1.7-fold in the striatum and threefold in the ventral mesencephalon, with corresponding elevations in calmodulin messenger RNAs. In response to the 10th dose of amphetamine, however, the degree of increase in calmodulin was diminished in the striatum and ablated in the ventral mesencephalon. Correspondingly, select species of calmodulin messenger RNA were decreased from control levels. In the frontal cortex or nucleus accumbens, calmodulin levels were basically unaltered by the first or 10th doses of amphetamine, but both calmodulin and its messenger RNA were altered with time upon cessation of the drug. Three days later, both calmodulin protein and messenger RNA were decreased in select brain areas. By seven days after the 10th injection, calmodulin content was altered compared to saline controls in all areas, but the change in messenger RNA no longer paralleled the change in protein.Our findings demonstrate that both calmodulin protein and select species of calmodulin messenger RNA are altered by acute amphetamine, but this effect is attenuated after repeated, intermittent amphetamine. There are further time-dependent changes after cessation of repeated amphetamine, which may reflect compensatory neuronal responses. The alterations in calmodulin content and synthesis could contribute to changes in patterns or duration of behaviors that occur upon cessation of repeated amphetamine.

Amphetamine↗
PubMed1987

A mutant Paramecium with a defective calcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation.

The Paramecium mutant, pantophobiac A, has a defect that results in an in vivo loss of calcium-dependent potassium efflux channel activity. This defect is corrected fully by the microinjection of wild-type Paramecium calmodulin into pantophobiac A cells and is partially restored by calmodulins from other organisms, but it cannot be restored by microinjection of pantophobiac calmodulin. Overall, these results suggested that wild-type Paramecium calmodulin has unique features that allow it to restore fully a normal phenotype and that the defect in pantophobiac A might be an altered calmodulin molecule. Previous studies established the amino acid sequence of wild-type calmodulin and showed that Paramecium calmodulin has several differences from other calmodulins, including the presence of dimethyllysine at residue 13. To test directly the possibility that calmodulin from the pantophobiac mutant might be altered, we purified the mutant calmodulin and compared its properties to those of wild-type Paramecium calmodulin. We found one amino acid sequence difference between the two Paramecium calmodulins: a phenylalanine in the mutant protein, instead of a serine, at residue 101. This change is at a calcium-liganding residue in the third calcium-binding loop. These and previous studies demonstrate that comparatively subtle changes in the structure of calmodulin can result in quantitative alterations in in vivo activity, provide insight into the in vivo roles of calmodulin and the regulation of ion channels, and demonstrate that functional alterations of calmodulin are not necessarily lethal.

Amino Acid Sequence↗
PubMed1986

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.

Animals↗