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PubMed1987

Calmodulin binding by calcineurin. Ligand-induced renaturation of protein immobilized on nitrocellulose.

The interaction of calmodulin with calcineurin, a calcium- and calmodulin-stimulated protein phosphatase, was investigated using a solid-phase assay. Binding of 125I-calmodulin by calcineurin immobilized on nitrocellulose membrane filters was of high affinity, reversible, and calcium-dependent. Complex binding kinetics reflected a time- and calcium/calmodulin-dependent conformational change of calcineurin which was shown to be ligand-induced renaturation. After renaturation and removal of calmodulin, immobilized calcineurin exhibited simple 125I-calmodulin binding kinetics with a single class of independent sites. The maximum stoichiometry of 125I-calmodulin binding to immobilized calcineurin was 0.1 mol/mol. The association rate (K1 = 8.9 x 10(3) M-1 S-1) and the dissociation rate (K-1 = 8.5 x 10(-5) s-1) yielded a dissociation constant of Kd = 10 nM. Equilibrium binding analyses gave a Kd value of 16 nM. The affinity of 125I-calmodulin for immobilized calcineurin was half that of unmodified calmodulin. Using equilibrium competition experiments, we determined, for the first time, the dissociation constant for the binding of native calmodulin by calcineurin in solution, Kd less than or equal to 0.1 nM (Kd for 125I-calmodulin = 0.23 +/- 0.09 nM). The effects of ionic strength and pH on 125I-calmodulin binding to immobilized calcineurin were characterized. The dissociation rate was dependent on free calcium concentration, with half-maximal rate at 700 nM calcium. 125I-Calmodulin equilibrium binding by the immobilized A subunit of calcineurin exhibited half the affinity of the holoenzyme, Kd = 30 nM. The described phenomenon, of reversible denaturation associated with immobilization of a protein on nitrocellulose, may be a general one open to exploitation in other systems.

Animals↗
PubMed1985

Selective affinity chromatography with calmodulin fragments coupled to sepharose.

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Characterization of a calmodulin-dependent protein phosphatase from human platelets.

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.

Animals↗
PubMed1994

Interaction of calmodulin with the cyclic GMP-gated channel of rod photoreceptor cells. Modulation of activity, affinity purification, and localization.

The cGMP-gated cation channel of rod photoreceptor cells plays a central role in the phototransduction process by controlling the influx of cations into the rod outer segment in response to changes in cGMP levels. Previous studies have shown that the cGMP-gated channel in native rod outer segment membrane vesicles is modulated by calmodulin in a calcium-dependent manner. In this study we report that the immunoaffinity-purified channel consisting of the 63-kDa alpha-subunit and a 240-kDa protein is also modulated by calmodulin when reconstituted into lipid vesicles. In the absence of calmodulin, the purified channel had an apparent Km of 33 microM and a Hill coefficient of 3.3 for cGMP-dependent efflux of Ca2+ from reconstituted lipid vesicles. In the presence of calmodulin, the Km increased to 44 microM without affecting the Hill coefficient or maximum velocity of ion efflux. Calmodulin modulation of the channel is inhibited by the calmodulin antagonist, mastoparan. In the absence of mastoparan, the half-maximum inhibition of channel activity (IC50) occurred at 1.85 +/- 0.25 nM calmodulin at a cGMP concentration of 12.5 microM; in the presence of mastoparan, the IC50 value increased to 20.3 +/- 3.8 nM calmodulin. Based on the strong, selective interaction of calmodulin with the channel, an efficient, general method has been developed to isolate functionally active cGMP-gated channels from mammalian and amphibian photoreceptor membranes. Calmodulin extraction studies, Western blotting, and channel activity measurements indicate that endogenous rod outer segment calmodulin modulates the activity of the channel through its binding to the 240-kDa protein. From these studies we conclude that the 240-kDa protein of the cGMP-gated channel is a major calmodulin target protein of rod outer segment membranes.

Animals↗
PubMed1993

Neocortex and hippocampus contain distinct distributions of calcium-calmodulin protein kinase II and GAP43 mRNA.

Calcium-calmodulin protein kinase II and GAP43 are two molecules which have been linked to synaptic plasticity. Localization of mRNA for these molecules identifies the neuronal populations which have the potential to utilize these mechanisms. General descriptions for calcium-calmodulin protein kinase II or GAP43 mRNA have been previously reported. In light of recent evidence that suggests that at some sites these two molecules may interact, we sought to determine the cortical distribution in detail, and to examine the extent of overlap between neuronal populations containing each mRNA. To this end we have used in situ hybridization techniques to study the distribution of calcium-calmodulin protein kinase II and GAP43 mRNA in adjacent sections of adult rat forebrain. Overall, the distribution patterns were distinct but partially overlapping. For both calcium-calmodulin protein kinase II and GAP43, mRNA levels were highest in hippocampus, allo- and neocortex, compared to moderate to low levels in striatum and thalamic nuclei. Within the heavily labeled regions certain populations expressed both calcium-calmodulin protein kinase II and GAP43 mRNA at high levels, while other populations were selective for calcium-calmodulin protein kinase II. In the hippocampus, the stratum pyramidale of CA1-3 expressed high levels of both calcium-calmodulin protein kinase II and GAP43 mRNA. Granule cells of the fascia dentata and the stratum radiatum of CA3 both contained moderate to high levels of calcium-calmodulin protein kinase II mRNA, but near background levels of GAP43 mRNA label. Within the neocortex, deep layers were distinguished from superficial layers by their lack of calcium-calmodulin protein kinase II mRNA expression within the neuropil, and the presence of GAP43 mRNA in neurons located in layer V and the deepest part of layer VI. Thus, layer V and deep layer VI neurons showed high levels of label for both GAP43 and calcium-calmodulin protein kinase II mRNA, while neurons of superficial layers contained only calcium-calmodulin protein kinase II mRNA. These markers differentiate neuronal populations which can also be distinguished on the basis of their ability to undergo specific forms of synaptic plasticity. These different forms of plasticity may be due in part to the laminar-specific patterns of GAP43 and calcium-calmodulin protein kinase II mRNA that we have described.

Animals↗
PubMed1994

Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion.

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca2+] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca2+, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca2+]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 microM and 90% at 7 microM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 microM trifluoperazine. Quantitative immunofluorescence using fluorescein-labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3-day bone-attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9 nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP-dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent Km for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated.

Acids↗
PubMed1993

Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides.

Calmodulin-dependent protein kinases such as myosin light chain kinase (MLCK), calmodulin kinase II, and phosphorylase kinase contain specific sequences responsible for binding calmodulin. These regions are known as calmodulin-binding domains and in many cases are contained within sequences that are short enough to be synthesized by solid-phase techniques. The ability to chemically-synthesize target enzyme calmodulin-binding domains has permitted the use of a variety of biophysical techniques to study the interactions between calmodulin and calmodulin-binding domain peptides. The work reviewed here describes the development and characterization of peptides based on the sequence of the calmodulin-binding domain of skeletal muscle myosin light chain kinase which were labeled with the fluorescent reagent, acrylodan. Data are presented demonstrating the use of fluorescently-labeled peptides to study various aspects of calmodulin-peptide interactions including binding affinity, stoichiometry, specificity, changes in peptide conformation, and thermal stability of the peptide-calmodulin complex. These data indicate the peptides exhibit many of the salient features seen with calmodulin-target enzyme interactions. The fluorescently-labeled peptides should thus serve as useful models for studying calmodulin-target enzyme interactions at the molecular level.

Amino Acid Sequence↗
PubMed1981

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations.

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.

Ca(2+) Mg(2+)-ATPase↗
PubMed1992

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.

Animals↗
PubMed1993

Binding of 3H-melatonin to calmodulin.

Studies in melatonin mechanism of action have suggested that one of them could be the binding of the hormone to calmodulin. We assessed calmodulin-melatonin binding by combining liposome incorporation of calmodulin with separation of free and bound 3H-Melatonin by a rapid ultrafiltration method. Specific binding to calmodulin was saturable, reversible, Ca(++)-dependent, ligand selective, and showed high affinity. Saturation as well as association-dissociation studies revealed that 3H-Melatonin binds to a single site on the calmodulin molecule with a Kd of 188 pM and a total binding capacity Bmax of 35 pM/ug of calmodulin. Displacement experiments showed that the relative order of potency of some compounds for inhibition of 3H-Melatonin was as follows: Melatonin > 6-chloromelatonin > 6-hydroxymelatonin > luzindole > trifluoperazine. The results explain our previously reported melatonin effects such as cytoskeletal rearrangements, inhibition of calmodulin dependent phosphodiesterase activity as well as the modification of Ca(++)-calmodulin electrophoretic mobility. The high affinity of melatonin binding to calmodulin suggests that the hormone is able to modulate cell activity by intracellularly binding to calmodulin at physiologically ranges. Melatonin-calmodulin binding could modulate many intracellular Ca++ functions and thus, the set-point for cell activity will follow the rhythmic circulating levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well preserved compounds, their interaction may represent a primary mechanism for both the regulation and the synchronization of cell physiology.

Animals↗
PubMed1985

Calmodulin regulation of adenylate cyclase activity.

Calmodulin-dependent stimulation of adenylate cyclase was initially thought to be a unique feature of neural tissues. In recent years evidence to the contrary has accumulated, calmodulin-dependent stimulation of adenylate cyclase now being demonstrated in a wide range of structurally unrelated tissues and species. Demonstration of the existence of calmodulin-dependent adenylate cyclase has in nearly all instances required the removal of endogenous calmodulin. It is not yet clear whether calmodulin-dependent and calmodulin-independent forms of the enzyme exist and whether some tissues (such as heart) lack a calmodulin-dependent adenylate cyclase. The presence of calmodulin appears largely responsible for the ability of the adenylate cyclase enzyme to be stimulated by submicromolar concentrations of calcium; it may not be relevant to the inhibition of the enzyme which occurs at higher concentrations of calcium. The physical relationship of calmodulin to the plasma membrane bound enzyme (or to the soluble forms of the enzyme) is not known nor is the mechanism of adenylate cyclase activation by calmodulin clear; current data suggest some involvement with both the N and C units of the enzyme. Finally, it is possible that in vivo calcium contributes to the duration of the hormone stimulated cyclic AMP signal. Thus current in vitro data suggest that optimal hormonal activation of calmodulin-dependent adenylate cyclase occurs at very low intracellular calcium concentrations, comparable to those found in the resting cell; conversely the enzyme is inhibited as intracellular calcium increases, following for example agonist stimulation of the cell. These higher calcium concentrations would then activate calmodulin-dependent phosphodiesterase. Such differential effects of calcium on adenylate cyclase and phosphodiesterase would ultimately restrict the duration of the hormone-induced cyclic AMP signal.

Adenylyl Cyclases↗
PubMed1990

Ontogenetic development of calmodulin mRNA in rat brain using in situ hybridization histochemistry.

An oligonucleotide probe complementary to the area on calmodulin coding for the calcium binding domain II on calmodulin was used to study the ontogenetic development of calmodulin mRNA in rat brain using in situ hybridization histochemistry. The hybridization signal for this probe was saturable, RNAse sensitive and was displaced by excess unlabelled calmodulin probe but was not displaced by an S-100 probe or by another calmodulin probe which was complementary to the mRNA coding for a different portion of calmodulin. At birth, high levels of calmodulin mRNA were found in hippocampus, cerebral cortex, thalamic nuclei and corpus striatum, and relatively low levels were in white matter. The rate at which calmodulin mRNA changed during development in the different brain areas varied with the brain area. At postnatal day one, the highest hybridization signals were in the cortical plate of the cerebral cortex, in thalamus and in the pyramidal cell layers of hippocampus and pyriform cortex. This distribution became more uniform with age. In contrast to most other brain areas, calmodulin mRNA in cerebellum increased markedly between one and 32 days postnatal; the hybridization signal was low at day one and was confined to the external germinal layer, but by day 16 calmodulin mRNA was largely in the granular layer. These results taken together with other findings on the effects of calmodulin on cellular growth differentiation, suggest that calmodulin may play a role in neuronal maturation.

Aging↗
PubMed1992

Cyclic AMP selectively up-regulates calmodulin genes I and II in PC12 cells.

Calmodulin is encoded by three genes in rat tissues, which collectively transcribe five different mRNAs. We showed previously that the pheochromocytoma cell line, PC12, expresses all five of these mRNAs and that treating PC12 cells with nerve growth factor differentially induces the calmodulin mRNAs; the greatest increase was seen by 24 h in the 1.4 kb transcript from calmodulin gene II. In the present study we found that treating PC12 cells with dibutyryl cyclic AMP also differentially increased the levels of the calmodulin mRNAs. However, dibutyryl cyclic AMP produced increases as early as 3 to 6 h, with the greatest increase (about 3-fold) being seen in the level of the 1.7 kb mRNA transcribed from calmodulin gene I. The transcripts of 4.1 kb (calmodulin gene I) and 1.4 kb (calmodulin gene II) were also increased, but the 2.3 kb transcript from calmodulin gene III remained stable. Another cyclic AMP analogue, chlorophenylthio cyclic AMP, produced effects similar to those of dibutyryl cyclic AMP, but dibutyryl cyclic GMP did not. Pretreatment with cycloheximide blocked the increase in the 4.1 kb calmodulin mRNA induced by dibutyryl cyclic AMP, but only partially blocked the increase in the 1.4 kb and 1.7 kb transcripts. Phorbol 12-myristate 13-acetate, which can induce some cyclic AMP responsive genes, failed to significantly change the levels of any of the calmodulin mRNAs. These studies show that, like nerve growth factor, cyclic AMP can selectively up-regulate the transcripts from calmodulin genes in PC12 cells, but the time-course and type of transcripts induced by cyclic AMP are distinct. These results suggest that the mechanisms by which these two agents alter the calmodulin transcripts are different.

Animals↗
PubMed1980

Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland.

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1981

Dynamics of calmodulin and cyclic AMP phosphodiesterase in plasma membranes of rat livers and ascites hepatomas.

1. Plasma membranes from ascites hepatoma cells (AH-7974, AH-130) contained much smaller amounts of calmodulin (about half) and cyclic AMP phosphodiesterase (about one-third) compared to plasma membranes of rat livers. 2. Some of calmodulin molecules in liver plasma membranes were released by repeated washing. The 'washed' liver plasma membranes showed the presence of specific binding sites for externally added calmodulin molecules (bovine brain) (N = 140 pmol/mg protein, Kd = 7.9 . 10(-8) M). The calmodulin content of AH-7974 plasma membranes was not reduced by repeated washing. The binding of calmodulin to the 'washed' AH-7974 plasma membranes was only of nonspecific nature with negative cooperativity. 3. Plasma membranes (liver and AH-7974) appeared to contain both calmodulin-dependent and calmodulin-independent phosphodiesterase, but the stimulation by externally added Ca2+ plus calmodulin was rather small. Externally added calmodulin-dependent phosphodiesterase (bovine brain) was bound more to 'washed' liver plasma membranes than to 'washed' AH-7974 plasma membranes. Newly bound phosphodiesterase appeared to be more sensitive to the stimulation by Ca2+ plus calmodulin in 'washed' hepatoma plasma membranes than in 'washed' liver plasma membranes. 4. Preincubation of 'washed' plasma membranes (liver and hepatoma) with calmodulin did not affect the binding of phosphodiesterase, but the sensitivity of phosphodiesterase to the stimulation by Ca2+ plus calmodulin in hepatoma plasma membranes was lost.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain.

Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.

Animals↗
PubMed1995

Anti-calmodulin potency of indol alkaloids in in vitro systems.

We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine, and navelbine) bind to calmodulin in a Ca(2+)-dependent manner. We designed direct binding tests (fluorescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 microM and 3 microM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC50 = 90 microM, 400 microM, and 430 microM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and vinblastine, similarly to trifluoperazine, the classic calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine possess identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.

Animals↗
PubMed1989

Glycation of calmodulin: chemistry and structural and functional consequences.

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗