Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Calmodulin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1983

Role of calmodulin in thyroid hormone stimulation in vitro of human erythrocyte Ca2+-ATPase activity.

Because human erythrocyte membrane Ca2+-ATPase is a calmodulin-dependent enzyme, and because physiological levels of thyroid hormone stimulate this enzyme system in vitro, we have studied the role of calmodulin in this model of extranuclear thyroid hormone action. Ca2+-ATPase activity in the absence of thyroid hormone ("basal activity") was increased by inclusion in the preassay incubation mixture of purified calmodulin or hypothyroid erythrocyte hemolysate that contained calmodulin (39 micrograms calmodulin/ml packed cells, determined by radioimmunoassay); addition of L-thyroxine or 3,5,3'-triiodo-L-thyronine (10(-10)M) significantly enhanced (P less than 0.001) enzyme activity in the presence of calmodulin or hemolysate. The stimulatory effects of thyroid hormone, calmodulin, and hemolysate were additive. At 5-10 microM, trifluoperazine, an antagonist of calmodulin, inhibited thyroid hormone stimulation of Ca2+-ATPase activity. Higher concentrations of trifluoperazine (50-100 microM) inhibited basal and hormone-stimulated enzyme activity, with or without added calmodulin. Anti-calmodulin antibody (10-50 micrograms antibody/mg membrane protein) inhibited basal, calmodulin-stimulated and thyroid hormone-stimulated Ca2+-ATPase activity. Membrane preparations were shown by radioimmunoassay to contain residual endogenous calmodulin (0.27 +/- 0.02 micrograms/mg membrane protein). The latter accounts for the effect of trifluoperazine and calmodulin antibody on membrane Ca2+-ATPase activity in the absence of added purified calmodulin. These results support the conclusion that the in vitro action of physiological levels of iodothyronines on human erythrocyte Ca2+-ATPase activity requires the presence of calmodulin.

Antibodies↗
PubMed1985

Antibodies to calmodulin during experimental Trypanosoma brucei rhodesiense infections in rabbits.

Calmodulin is an intracellular Ca2+ receptor protein which regulates a wide variety of enzymatic processes in eukaryotic cells examined in detail. Native calmodulin is not antigenic in rabbits because of its small size, high degree of amino acid sequence conservation and hydrophobicity. African trypanosomes contain a novel calmodulin which is structurally distinct from bovine brain and Tetrahymena calmodulins. In the present study, we examine the antibody response towards these calmodulins during chronic Trypanosoma brucei rhodesiense infections. Injection of purified trypanosome calmodulin into rabbits stimulates the production of specific IgG antibodies which recognize trypanosome, but not bovine brain or Tetrahymena calmodulins. By contrast, during chronic T. brucei infections in rabbits, antibodies (IgG + IgM + IgA) that recognize trypanosome, Tetrahymena and mammalian calmodulins arise. When only IgG antibodies are evaluated from infection sera, the major response is against mammalian and Tetrahymena calmodulins. Significantly fewer IgG antibodies are measured in the infection sera which recognize trypanosome calmodulin, while the non-specific control protein, chicken ovalbumin, is not recognized. Peak IgG antibody responses against calmodulin occur between Days 30-34 post-infection. Competition assays indicate that Tetrahymena and mammalian calmodulins are recognized at identical epitopes which are distinct from epitopes on trypanosome calmodulin. We conclude that, in the context of chronic T. brucei infections in rabbits, antibodies arise which are able to recognize mammalian host calmodulin.

Animals↗
PubMed1985

Metal ions as allosteric regulators of calmodulin.

Previously we have shown that the fluorescence of the dihydropyridine calcium antagonist felodipine provides an accurate means of monitoring the formation of an allosterically potentiated conformer of calmodulin (Mills, J. S., and Johnson, J. D., (1985) Biochemistry 24, 4897-4903). Characteristic of this conformer is the abolition of cooperativity among the two felodipine-binding sites on calmodulin and a 20-fold increase in the apparent affinity of calmodulin for felodipine. In the present study, we find that the metal cations La3+, Tb3+, Pb2+, and Cd2+ are all capable of abolishing the cooperativity (Hill coefficient = 2.0) among the two felodipine-binding sites on calmodulin and can increase the apparent affinity of calmodulin for felodipine by approximately 20-fold. These effects are seen either in the presence or absence of calcium and are half-maximal at 8, 12, 22, and 1000 microM, respectively. Zinc and H+ are capable of producing similar potentiations of felodipine binding (half-maximal at 570 microM, and pH 5.8), but only in the presence of calcium. In each case, the calcium-binding sites of calmodulin must be occupied (by calcium, La3+, Tb3+, Pb2+, or by Cd2+) before these metals can bind to sites which are distinct from the calcium-binding sites to produce the active conformer of calmodulin which exhibits enhanced affinity for felodipine. Mercury and copper can compete with these potentiating metal cations on calmodulin and produce an inactivation of this active calmodulin conformer. These studies suggest that some metals including La3+, Tb3+, Pb2+, Cd2+, Zn2+ and protons are capable of binding to a calcium-calmodulin complex and forming an allosterically active species of calmodulin which cannot be maintained by physiological concentrations of calcium ions alone. Mercury and copper, on the other hand, are capable of inactivating this active calmodulin conformer independent of the presence of calcium on calmodulin. These findings are examined in terms of the mechanism of action of calmodulin and its possible role in heavy metal toxicity.

Calcium↗
PubMed1984

Carboxylmethylation of phosphodiesterase attenuates its activation by ca2+-calmodulin.

Carboxylmethylation of several preparations of cAMP phosphodiesterase by the enzyme protein O- carboxylmethyltransferase and S-adenosylmethionine reduces the extent to which the enzyme was activated by native calmodulin. In contrast, carboxylmethylation of calmodulin produced only a slight reduction in the ability of calmodulin to activate cAMP phosphodiesterase. The effect of carboxylmethylation of calmodulin was most prominent at subsaturating calmodulin concentrations, whereas the reduction in the activation of carboxylmethylated cAMP phosphodiesterase was independent of calmodulin concentration. Kinetics and stoichiometric analysis of calmodulin carboxylmethylation indicated that less than 5% of calmodulin was methylated and that the Km of protein O- carboxylmethyltransferase for calmodulin was approximately 350 microM. The extent of calmodulin carboxylmethylation was not affected by either EGTA or Ca2+. When homogeneous bovine brain phosphodiesterase was carboxylmethylated , a rapid decrease in calmodulin-induced stimulation was noted, occurring within 30 s of incubation. Acidic sodium dodecyl sulfate-gel electrophoresis of bovine brain phosphodiesterase revealed a major band of 60,000 daltons which contained radio-activity after carboxylmethylation . Stoichiometric analysis revealed that approximately 20% of the phosphodiesterase was carboxylmethylated . Thus, although calmodulin can serve as a substrate for carboxylmethylation , it appears that carboxylmethylation has a greater effect on calmodulin-dependent phosphodiesterase activity when the target enzyme, rather than calmodulin, is carboxylmethylated .

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

Insulin-dependent phosphorylation of calmodulin in rat hepatocytes.

Insulin-stimulated phosphorylation of calmodulin in vivo was examined using a highly specific anti-calmodulin monoclonal antibody combined with high resolution two-dimensional gel electrophoresis. The two major isoforms of calmodulin immunoprecipitated from insulin-treated hepatocytes migrated on two-dimensional gel electrophoresis to the same position as nonphosphorylated calmodulin and calmodulin phosphorylated in vitro. Immunoblotting verified the identity of calmodulin. Insulin enhanced the phosphorylation of calmodulin 3.1 +/- 0.4-fold (mean +/- S.E., n = 10), with a stoichiometry in insulin-treated hepatocytes of 0.47 +/- 0.06 (mean +/- S.E., n = 3) mol of phosphate/mol of calmodulin. Two-dimensional phosphopeptide mapping of calmodulin immunoprecipitated from rat hepatocytes and calmodulin phosphorylated in vitro by the insulin receptor kinase or casein kinase II revealed several common phosphopeptides. The common phosphopeptides that appeared insulin-sensitive in intact cells comprised 61 and 40% of casein kinase II- and insulin receptor-catalyzed 32P incorporation into calmodulin in vitro, respectively. This suggests that casein kinase II and the insulin receptor kinase are, at least in part, responsible for insulin-stimulated phosphorylation of calmodulin in rat hepatocytes. These data indicate that phosphorylation of calmodulin in intact hepatocytes is significantly enhanced by insulin, supporting a critical role for calmodulin in insulin signal transduction.

Animals↗
PubMed1994

Gain of function mutations for yeast calmodulin and calcium dependent regulation of protein kinase activity.

Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence. However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity. Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact. An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin. These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.

Amino Acid Sequence↗
PubMed1996

Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin.

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.

Adenosine Triphosphatases↗
PubMed1989

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella.

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1996

Haloperidol and MK-801 block increases in striatal calmodulin resulting from repeated amphetamine treatment.

Repeated, intermittent treatment with amphetamine leads to a behavioral sensitization characterized in rats by an increase in locomotor activity and a more rapid onset of stereotyped behaviors. Induction of amphetamine sensitization is blocked by dopamine and N-methyl-D-aspartate (NMDA) antagonists. We have reported an increase in the content of the Ca2(+)-binding protein, calmodulin, in striatum and limbic forebrains from rats given repeated, intermittent amphetamine. To determine whether the increase was related to development of amphetamine sensitization, we examined whether the increase in calmodulin would be blocked by the dopamine antagonist, haloperidol, or the NMDA antagonist, MK-801. Rats were given amphetamine or saline twice weekly for 5 weeks. Thirty min prior to the amphetamine, rats were pretreated with 0.25 mg/kg haloperidol s.c., 0.1 mg/kg MK-801 i.p. or saline. Twice weekly amphetamine treatment increased calmodulin in the cytosol fraction of striatum and limbic forebrain and the increase was blocked by pretreatment with either haloperidol or MK-801. Neither antagonist alone affected cytosolic calmodulin. Haloperidol pretreatment, but not amphetamine or MK-801, increased calmodulin in striatal but not limbic forebrain membranes. Calmodulin-binding proteins were examined by biotinylated calmodulin blotting to determine if repeated, intermittent amphetamine altered the content of calmodulin-binding proteins in striatal cytosol or membranes. A band of 73 kDa was increased in striatal membranes. Immunoblotting with antisera to caldesmon, a cytoskeletal calmodulin-binding protein of 77 kDa, demonstrated increases in immunoreactivity in striatal membranes and cytosol. These data suggest that dopaminergic and glutamatergic components are required for the increases in striatal and limbic forebrain calmodulin and that the rise in calmodulin is related to the development of amphetamine sensitization. In addition, the content of select calmodulin-binding proteins can be coordinately regulated with increases in calmodulin.

Amphetamine↗
PubMed1996

Effects of cell culture time and bone matrix exposure on calmodulin content and ATP-dependent cell membrane acid transport in avian osteoclasts and macrophages.

Osteoclasts mediate bone resorption by secretion at the site of bone attachment. This process depends on calmodulin concentrated at a specialized acid-secreting membrane. We hypothesized that increased calmodulin and bone attachment were required for acid secretion. We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells. Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone. Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake. Calmodulin decreased in osteoclasts cultured without bone. Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days. Bone had no effect. Calmodulin mRNA was similar in osteoclasts with or without bone. However, only osteoclasts cultured with bone retained acid transport capacity. Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production. Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect. Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism. In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro. Increased calmodulin correlated with membrane acid transport capacity in both cell types. The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.

Animals↗
PubMed1994

Tyrosine-phosphorylated calmodulin has reduced biological activity.

Calmodulin is phosphorylated by the purified insulin receptor on tyrosine residues with a maximum stoichiometry of 1 mol phosphate/mol of calmodulin. Isolated tryptic phosphopeptides were sequenced by manual Edman degradation and demonstrated that calmodulin is equally phosphorylated on tyrosine 99 and tyrosine 138. Phosphorylated calmodulin has a decreased affinity (K0.5 = 4.2 nM) for the 63-kDa isozyme of cyclic nucleotide phosphodiesterase compared to nonphosphorylated calmodulin (K0.5 = 2.1 nM). The K0.5 for Ca2+ is marginally increased from 2.8 to 3.2 microM in the presence of phosphotyrosyl calmodulin. The effect of the calmodulin antagonist, mastoparan, was investigated to determine whether mastoparan would differentially inhibit calmodulin- or phosphocalmodulin-dependent enzyme activity. The IC50 of mastoparan is fourfold lower for phosphotyrosyl calmodulin compared to nonphosphorylated calmodulin. Phosphorylation of calmodulin may provide a mechanism for the differential regulation of calmodulin-dependent enzymes. These observations further support a potentially important regulatory function of calmodulin phosphorylation in signal transduction.

Amino Acid Sequence↗
PubMed1988

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.

Adenosine Triphosphate↗
PubMed1982

Interaction of drugs with calmodulin. Biochemical, pharmacological and clinical implications.

Calmodulin is a widely distributed, highly active, calcium-binding protein that influences a number of important biological events. Accordingly, agents that inhibit the activity of calmodulin should have profound pharmacological effects. Within the past few years, a number of compounds have been identified that inhibit calmodulin. The most potent of these described so far include certain antipsychotic drugs, smooth muscle relaxants, alpha-adrenergic blocking agents and neuropeptides. Studies of the physicochemical and structural properties of a variety of calmodulin inhibitors have shown that there are ionic and hydrophobic interactions between the drug and calmodulin. From the limited studies conducted so far, we conclude that, for a compound to inhibit calmodulin, it should carry a positive charge at physiological pH, presumably to interact with negative charges on the highly acidic calmodulin, and have hydrophobic groups, presumably to interact with lipophilic regions on calmodulin. But these two factors are not the only ones that are involved in inhibiting calmodulin, for many highly charged and highly hydrophobic agents have relatively little effect on calmodulin activity. The structural relationships between these ionic and hydrophobic regions and other, as yet identified, factors are also important. Many of the biochemical actions of the phenothiazine antipsychotic agents can be explained by the common mechanism of their binding to, and inhibiting, calmodulin. The question of whether these biochemical actions can explain their pharmacological and clinical effects is still unclear. The fundamental role calmodulin plays in biology suggests that this calcium binding protein may provide a new site for the pharmacological manipulation of biological activity. The calmodulin inhibitors described thus far hardly scratch the surface of this fertile area of research.

Antipsychotic Agents↗
PubMed1988

Characteristics of the binding of phenoxybenzamine to calmodulin.

To determine the factors that influence the interaction between phenoxybenzamine and calmodulin, the binding of phenoxybenzamine to calmodulin was determined by equilibrium dialysis under a variety of experimental conditions. This interaction was found to be similar in some respects to the interaction between phenothiazines and calmodulin. It was saturable, with between 1 and 2 mol of phenoxybenzamine bound to 1 mol of calmodulin. It was also dependent upon temperature, the presence of a divalent cation such as calcium, and on pH, showing maximum binding at pH 6.5 with little binding at pH values below 4.2 or above 8.0. The site at which phenoxybenzamine bound to calmodulin appears to be similar to that at which certain antipsychotic agents bind, since several of them, including penfluridol, pimozide and spiroperidol, prevented the binding of phenoxybenzamine to calmodulin. However, in contrast to the reversible binding of most phenothiazines to calmodulin, phenoxybenzamine bound to calmodulin irreversibly. The binding of phenoxybenzamine to calmodulin was fairly selective in that other alpha-adrenergic agents such as prazosin, yohimbine and clonidine failed to bind to calmodulin when examined under the same experimental conditions. In addition, phenoxybenzamine showed little or no calcium-dependent binding to the S-100 protein, bovine serum albumin or cytochrome c. The irreversible complex between phenoxybenzamine and calmodulin may be useful for inhibiting certain calmodulin-dependent reactions and for studying the various biological functions of calmodulin.

Calmodulin↗
PubMed1994

Calmodulin binding proteins in the membrane vesicles released during the acrosome reaction and in the perinuclear material in isolated acrosome reacted sperm heads.

Calmodulin has been suggested as the Ca(2+)-mediator in diverse cellular functions via its interaction with a number of proteins in a calcium-dependent manner. Its participation in the acrosome reaction has been suggested based on its localization in the acrosome region, on the effects produced by calmodulin antagonists, and by the changes in calmodulin compartmentation observed to occur throughout guinea pig acrosome reaction. To define the role of calmodulin in the membrane fusion events that occur during the acrosome reaction, the identification of calmodulin-binding proteins, by the overlay technique with biotinylated or unmodified calmodulin, was made in the following sperm fractions: in the membrane vesicles released during the acrosome reaction, in the remaining perinuclear material of acrosome reacted sperm heads and in a total membrane fraction from intact spermatozoa. The membrane vesicles released after the acrosome reaction showed four major calmodulin-binding proteins, M(r)s 66, 95, 97 and 110 kDa. The perinuclear material showed a 31-34, 43 and 97 kDa calmodulin-binding polypeptides. The membrane fraction from intact sperm showed eleven calmodulin-binding proteins, M(r)s between 14-110 kDa. Most of the binding proteins detected by this method corresponded to the class of calcium-independent calmodulin-binding proteins but proteins which only interacted with calmodulin in a calcium-inhibited mode were also observed. No calcium-dependent calmodulin-binding proteins were detected in any of the fractions studied. A possible role of these binding proteins in calmodulin compartmentation is discussed. The potential role of these binding proteins in membrane fusion and in membrane receptor localization in the postacrosomal region remain to be defined.

Acrosome↗
PubMed1985

Comparative structural analysis of calmodulins from Trypanosoma brucei, T. congolense, T. vivax, Tetrahymena thermophila and bovine brain.

Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals. The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin. Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain. Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures. These same analyses do not distinguish trypanosome calmodulins from each other. Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins. Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine. All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium. These data demonstrate the extent to which functional calmodulins vary in their structures. We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.

Animals↗
PubMed1985

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver.

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.

Adenylyl Imidodiphosphate↗
PubMed2002

Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca(2+)-dependent [125I]Calmodulin binding.

Constitutive isoforms of nitric oxide synthase (NOS) are activated by transient binding of Ca(2+)/Calmodulin. Here, we characterize the binding of Calmodulin to purified neuronal NOS (nNOS). [125I]Calmodulin bound to a single class of non-interacting and high affinity sites on nNOS. [125I]Calmodulin binding achieved rapid saturation, was linear with nNOS concentration, and exhibited a strict dependence on [Ca(2+)]. Neither affinity nor extent of [125I]Calmodulin binding was affected by L-arginine, NADPH or Tetrahydrobiopterin. Native Calmodulin and engineered Calmodulin homologs [i.e., duplicated N-terminal (CaMNN)] potently displaced [125I]Calmodulin. CaMNN supported nNOS catalysis, but required approximately five-fold more Ca(2+) for comparable activity with native Calmodulin. Taken with results from kinetic analyses of [125I]Calmodulin association and dissociation, our findings suggest four sequential steps in activation of nNOS by Calmodulin: (1) Ca(2+) binds to Calmodulin's C-lobe, (2) the C-lobe of Calmodulin binds NOS, (3) Ca(2+) binds to the N-lobe of Calmodulin, and (4) the N-lobe binds to nNOS. Activation of nNOS only occurs after completion of step (4), with the displacement of nNOS's autoinhibitory insert. Upon intracellular Ca(2+) sequestration, deactivation of nNOS would proceed in reverse order.

Animals↗