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PubMed1992

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.

Amino Acid Sequence↗

PubMed1983

Compound 48/80 is a selective and powerful inhibitor of calmodulin-regulated functions.

Compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 48/80 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 micrograms/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 micrograms/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound 48/80 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound 48/80 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 48/80 bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 48/80 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound 48/80 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 48/80, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound 48/80 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 48/80 is proposed to be a promising tool for studying calmodulin-dependent processes.

Animals↗

PubMed1984

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on the rate of calmodulin binding to erythrocyte Ca2+-ATPase.

The erythrocyte Ca2+-ATPase shifts reversibly between two states, the calmodulin-deficient A-state and the calmodulin-saturated B-state, dependent on calcium and calmodulin. The effects on this system of the four drugs, trifluoperazine, compound 48/80, TMB-8 and verapamil were studied. All four drugs inhibited the maximum activity of the B -state Ca2+-ATPase and, in addition, trifluoperazine and compound 48/80 in higher doses inhibited the A-state. Furthermore, the four drugs decreased the calmodulin sensitivity of the Ca2+-ATPase in the order of decreasing effect: trifluoperazine greater than compound 48/80 greater than TMB-8 greater than verapamil. In the same order of decreasing effect the drugs increased the time required for full calmodulin activation of the A-state of Ca2+-ATPase, whereas the drugs had only small effects on the rate of deactivation of the B-state, caused by dissociation of calmodulin from the enzyme. It is discussed whether the effects on calmodulin activation were caused by a reduction of free calmodulin due to the formation of drug-calmodulin complexes or whether the drugs, especially trifluoperazine, compound 48/80 and TMB-8, by binding to the Ca2+-ATPase, decreased the rate constants for association of calmodulin and enzyme.

Calcium Channel Blockers↗

PubMed1989

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80.

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.

Biological Transport, Active↗

PubMed1990

Expression of calmodulin gene is down-regulated in diabetic BB rats.

In past studies, we have demonstrated that in streptozotocin-induced diabetic or spontaneously diabetic (BB) animal models, low Km cAMP phosphodiesterase and calmodulin are decreased while a low MW inhibitor of calmodulin is increased. To extend these studies, we have determined the rate of [35S]-methionine incorporation into calmodulin in isolated fat cells from these diabetic animals, i.e. streptozotocin-induced diabetic and the BB rats, spontaneous diabetic rat, non-diabetic rat, and control. We found markedly decreased rates of synthesis of calmodulin in the fully diabetic BB rat. In order to investigate the mechanism of the reduced calmodulin biosynthesis, we probed poly A+ mRNA from control and diabetic rat livers with a calmodulin specific anti-sense oligonucleotide probe and found that the fully diabetic animals, streptozotocin-induced diabetic and genetically diabetic BB, contained markedly reduced levels of calmodulin transcripts. Thus, both calmodulin protein and its putative mRNA are decreased in diabetic rat liver. We believe that in uncontrolled diabetes, the observed elevation in the levels of cyclic AMP in plasma and tissue results in part from decreased activity of phosphodiesterase. The insulin-sensitive phosphodiesterase appears to be regulated by calmodulin. We hypothesize that cyclic AMP phosphodiesterase inactivation in diabetes results in part from insulin insufficiency and to a less well-defined genetic lesion leading to calmodulin down-regulation.

Actins↗

PubMed1984

Increase of calmodulin-stimulated striatal particulate phosphorylation response in chronic haloperidol-treated rats.

Calcium- and calmodulin-dependent protein kinase and phosphatase activities were studied in rat striatal particulate preparations. The effect of Ca2+ (0.1-0.5 mM) on phosphorylation was completely abolished in the preparation which had been washed 3 times with a buffer containing ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA, 1.2 mM). Ca2+-stimulated phosphorylation was restored in a dose-dependent manner after calmodulin (1 microgram) was added to the assays. Ca2+ and calmodulin promoted the phosphate incorporation into two major striatal protein bands with estimated Mr at 52 and 40 kdaltons. The presence of phosphatase in the EGTA-pretreated preparations was negligible. Chronic treatment in rats with haloperidol (1 mg/kg, 20 days) produced a significant decrease in the Ca2+-independent phosphorylation but an increase in the extent of Ca2+ and calmodulin-dependent phosphorylation in the striatum. The chronic haloperidol treatment did not alter the striatal [125I]calmodulin binding curve. In vitro, haloperidol (even at 10(-4) M) had no effect on calmodulin-dependent phosphorylation. Haloperidol (10(-4) M) did not reduce the number but decreased the rate of [125I]calmodulin bindign to the striatal particulates. These data suggest that the link between dopamine receptors and calmodulin-dependent enzyme is dissociated in vitro. On the other hand, the potentiated sensitivity of calmodulin-dependent protein kinase in the chronic haloperidol-treated rats correlated with the supersensitive dopamine receptor responses occurred in these rats. Therefore, calmodulin-dependent protein kinase may biochemically regulate dopamine receptor functions in the striatum in vivo.

Animals↗

PubMed1985

Genotypic variation in striatal calmodulin content.

CBA/J and BALB/cJ mice have quantitative differences in the nigrostriatal projection. The number of nigral tyrosine hydroxylase reactive neurons, nigral and striatal tyrosine hydroxylase activity and the density of striatal D-2 dopamine receptors are all less in the CBA/J compared to the BALB/cJ mouse. An unrelated strain, the C57BL/6J, has a striatal D-2 dopamine receptor density that is intermediate to that of CBA/J and BALB/cJ mice. CBA/J mice also show deficits in the ability of brain monoaminergic receptor systems to develop supersensitivity. Calmodulin may participate in several striatal dopaminergic receptor mechanisms. Thus, striatal calmodulin was examined in CBA/J, C57BL/6J and BALB/cJ mice. Striatal calmodulin was greater in CBA/J mice than in C57BL/6J or BALB/cJ. In all three strains, cerebral cortical calmodulin was similar. The percent distribution of total striatal calmodulin between soluble and particulate fractions was similar in the three strains. Calcium redistributed soluble striatal calmodulin into the particulate fraction and EGTA shifted calmodulin from the particulate into the soluble fraction. The percent of total striatal calmodulin redistributed by either treatment was similar in all three strains. Gel filtration chromatography of heat-treated soluble extracts from CBA/J and BALB/cJ striatum was similar in elution pattern, although more calmodulin was observed in extracts from the CBA/J. Possible mechanisms for the strain differences in calmodulin are discussed along with their relationship to strain differences in striatal dopamine receptor subtypes.

Animals↗

PubMed1986

Cell cycle dependence of calmodulin levels during HL-60 proliferation and myeloid differentiation. No changes during pre-commitment.

The putative role of Ca2+ and calmodulin in regulating cell proliferation and differentiation was tested in HL-60 human promyelocytic leukemia cells. The dependence of retinoic acid (RA)-induced terminal myeloid differentiation of HL-60 promyelocytic leukemia cells on calmodulin levels and calcium ion flux was ascertained. RA-treated and untreated control cells were stained for cellular DNA with a Hoechst dye. Populations of G1/0, S and G2 + M phase cells were isolated by fluorescence activated cell sorting (FACS). Cytosolic calmodulin levels were then measured as a function of cell cycle phase for RA-treated and untreated cells using a radioimmunoassay. RA-treated cells were measured at early times, corresponding to the pre-commitment state, and late times, when significant cell differentiation had occurred. Cellular calmodulin levels increased with progression through the cell cycle. In contrast, no difference in calmodulin levels was observed between RA-untreated or -treated cells in the same cell cycle phases at early or late times. RA-induced HL-60 terminal myeloid differentiation was thus apparently not regulated by cellular cytosolic calmodulin levels. These conclusions were supported by the effects of calmodulin antagonists and calcium flux inhibitors. The calmodulin antagonists trifluoperazine and compound 48/80 both retarded cell growth in a concentration-dependent manner. But at concentrations where cellular effect was evidenced by slight growth inhibition, neither antagonist inhibited RA-induced cell differentiation or G1/0 growth arrest. The same was true of the gated calcium channel inhibitors, verapamil and nitrendipene, and the passive calcium flux inhibitor, CoCl2. RA-induced HL-60 cell differentiation and arrest in G0 was thus apparently not strongly dependent on cellular calmodulin levels or calcium flux. This is in strong contrast to murine erythroleukemia cells. The results argue against a central regulatory role for calmodulin or calcium flux in control of HL-60 growth arrest or differentiation.

Calmodulin↗

PubMed1984

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction.

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.

Adenosine Triphosphate↗

PubMed1989

Calmodulin-binding proteins in subcellular fractions of zones of the adrenal cortex.

The guinea pig adrenal cortex consists of a steroidogenic ACTH-responsive outer zone and an ACTH-unresponsive inner zone. It has been suggested that calmodulin plays an important role in ACTH-stimulated steroidogenesis. Thus, in an effort to examine the calmodulin 'system' in the guinea pig adrenal cortex model, Ca2+-dependent binding of calmodulin to proteins in subcellular fractions of the outer and inner zones was examined by the [125I]iodocalmodulin overlay technique and compared to similar studies utilizing pancreas, brain and liver tissue. Although the general pattern of calmodulin-binding proteins was similar for the two adrenocortical zones, quantitatively there was a striking difference with greater binding in the outer zone; this was particularly noteworthy for the mitochondrial fraction. The two most prominent calmodulin-binding proteins isolated from cytosol by calmodulin-Sepharose column chromatography had Mr of 60,000 and 47,000. The size of these two proteins suggested the presence of Ca2+/calmodulin-dependent protein kinase II. Western blot analysis, however, failed to demonstrate calmodulin kinase II in either zone, although it was clearly detectable in brain cytosol. The 60 K calmodulin-binding protein in the adrenal cortex also suggested the presence of the calmodulin-binding A subunit of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin. Western blot analysis did reveal the presence of calcineurin in the outer adrenocortical zone; it was not detectable, however, in the inner adrenocortical zone. The relation between the striking zonal differential for calmodulin-binding proteins and the zonal differential in ACTH-stimulated steroidogenesis in the guinea pig adrenal cortex will require further investigation.

Adrenal Cortex↗

PubMed1988

Calcium, calmodulin and cell proliferation.

Calcium and calmodulin have been proposed to be regulatory factors in cell cycle progression. Clonal mouse cell lines harboring episomally-carried genes have been prepared to address this question. Some lines produce extra calmodulin, others express antisense RNA to decrease calmodulin, while others produce the Ca2+-buffering protein parvalbumin. The results show that calmodulin acts at two points in the cell cycle--the G1/S boundary and metaphase transition. An additional Ca2+ event that is calmodulin-independent occurs at mitotic prophase. The elevated (or depressed) level of intracellular Ca2+ binding protein does not markedly affect gene expression. In cells containing excess calmodulin, the synthesis mechanisms that normally control the level of calmodulin post-transcriptionally are overridden. Genes normally expressed in G1 whose products are involved in growth control show increases in calmodulin over producing cell lines. Elevated calmodulin decreases tubulin mRNA presumably due to its effect on microtubule stability. The availability of cell lines in which calmodulin can be inducibly increased or decreased should provide tools to elucidate the molecular mechanisms that govern the regulatory roles for this protein in cell cycle progression.

Animals↗

PubMed1994

Protein kinase C regulates calmodulin expression in NRK cells activated to proliferate from quiescence.

We have investigated the levels of calmodulin protein and calmodulin mRNA species during proliferative activation of NRK cells. Cells activated to proliferate from quiescence started to replicate DNA at 15 h, reaching a maximum at 20 h after serum addition. The maximum of mitosis was observed at 24 h. Quiescent cells showed a calmodulin concentration of 1.5 ng/micrograms of protein. At 10 h after serum addition the amount of calmodulin started to increase, reaching values of 3.0 ng/micrograms of protein at 24 h. NRK cells expressed predominantly 3 species of calmodulin transcripts: the 1.7 kb from CaM I, the 1.4 kb from CaM II and the 2.3 kb from CaM III. The amount of all the 3 transcripts was low in quiescent cells and 10 h after activation the levels were already high, reaching a maximum around 20 h. At the latter time the amount of the 3 calmodulin mRNAs was 5-10-fold higher than in serum starved cells. Run-on experiments showed that at 20 h after activation the transcription rates of the 3 calmodulin genes were higher than in quiescent cells. The addition of protein kinase C inhibitors to the cultures blocked the increase of the calmodulin transcripts while inhibitors of protein kinase A did not have any effect. Moreover, the addition of submitogenic doses of phorbol 12-tetradecanoate induced the increase of all 3 calmodulin transcripts. These results indicate that protein kinase C regulates calmodulin expression when NRK cells are activated to proliferate.

Alkaloids↗

PubMed1986

Changes in conformation of spin-labeled calmodulin by phospholipids.

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.

Binding Sites↗

PubMed1993

Identification and characterization of nuclear calmodulin-binding proteins of Saccharomyces cerevisiae.

Nuclear calmodulin-binding proteins of the yeast Saccharomyces cerevisiae were investigated. The soluble fractions after serial treatments of the isolated nuclei with buffers containing the nonionic detergent NP-40 (F1), 0.5 M KCl (F2) and 2.0 M KCl (F3) in this order, and the residual proteins (F4) were obtained. The calmodulin-binding proteins of the nucleus and nuclear subfractions were identified using the gel overlay method using 125I-calmodulin. Each subnuclear fraction contained a large number of components that bound calmodulin in a Ca(2+)-dependent or -independent manners. The calmodulin-binding proteins were isolated from F1 and F2 subnuclear fractions by affinity chromatography. The affinity-purified proteins bound calmodulin in a Ca(2+)-dependent manner when analyzed using the gel overlay method. The major calmodulin-binding components of F1 were 44, 42, 36, 32 and 29 kDa proteins, and those of F2 were 200, 100, 40, 42, 36, 34 and 32 kDa proteins. The isolated proteins also contained several Coomassie-blue stained proteins that did not bind calmodulin and, therefore, may represent the proteins associated with the calmodulin-binding proteins. Antisera raised against the affinity-purified preparation of F1 and F2 recognized almost all of the calmodulin-binding proteins present in the fraction and several other proteins of the nucleus. The presence of Ca(2+)-dependent protein phosphatase (type 2B) in the nucleus was demonstrated by Western blotting. The enzyme was localized predominantly in F1 and F4.

Calmodulin-Binding Proteins↗

PubMed1994

Calmodulin, calbindin-D28K and calretinin in rat and chicken pineal glands: immunocytochemical and immunoblotting analysis.

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacological studies using calmodulin antagonists suggested that melatonin synthesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepancy, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants and Triton X-100-treated pellets denatured by sodium dodecyl sulfate, subjected to electrophoresis and immunoblotting using anti-calmodulin antibodies. Two different IgG (#465 and #860) purified from anti-calmodulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmodulin could also be detected in the Triton-treated pellet fractions, but no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, in addition to a calmodulin immunoreactive band, two other proteins with approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detected using anti-calmodulin #465. For comparison, similar immunoblot experiments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pellets revealed additional bands cross-reacting with each antibody. Anti-calbindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted strongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa protein.

Animals↗

PubMed1989

Calmodulin modulates thymocyte adenylate cyclase activity through the guanine nucleotide regulatory unit.

We have previously demonstrated in rat thymocyte plasma membranes that adenylate cyclase activity and its stimulation by 3,5,3'-triiodothyronine (T3) are influenced by calmodulin, and that these effects of calmodulin require calcium. In the present study, the mechanism by which calmodulin exerts its action was examined, in situ, in fresh plasma membranes isolated from rat thymocytes. Adenylate cyclase activity was potentiated by guanyl nucleotides, NaF and forskolin. Calmodulin did not affect basal adenylate cyclase activity. However, calmodulin influenced the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity, but had no effect on the fluoride-stimulated enzyme activity. This was evident from experiments with inhibitors of calmodulin: trifluoperazine, calmidazolium, and antibodies against calmodulin. The three inhibitors did not change basal adenylate cyclase activity, but all produced a marked decrease in the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity. The inhibitory effect of all three agents was reversed completely by the addition of calmodulin. The three inhibitors, however, failed to affect the fluoride-stimulated adenylate cyclase activity. In addition, T3, like the calmodulin inhibitors, did not change basal adenylate cyclase activity, increased the guanyl nucleotide- and forskolin-stimulated enzyme activity, but had no effect on the fluoride-stimulated enzyme activity. From these results I suggest that in the rat thymocyte calmodulin activation, and thereby T3 stimulation of the calcium-sensitive adenylate cyclase system is mediated through the guanine nucleotide regulatory unit.

Adenylyl Cyclases↗

PubMed1994

Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia.

Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a Km for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 microM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca2+, Mg2+, Mn2+, and Ni2+ stimulated calmodulin N-methyltransferase activity but Zn2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory but W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.

Animals↗

PubMed1990

Evidence for a specific recognition site for tiflucarbine on calmodulin.

The putative antidepressant drug tiflucarbine (BAY P 4495) has previously been shown to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase competitively with respect to calmodulin. In order to determine whether this effect is mediated by a direct interaction with calmodulin, we measured the effects of radiolabelled triflucarbine in a direct ligand binding assay, using agarose-immobilized calmodulin. [3H]Tiflucarbine associated with low micromolar affinity with an apparently homogeneous class of recognition sites on calmodulin-agarose. No binding could be observed on calmodulin-deficient agarose. The effect was specific, saturable and reversible. Triflucarbine was the most potent calmodulin antagonist from a variety of structural analogues examined. The potencies of these derivatives to inhibit calmodulin-stimulated phosphodiesterase significantly correlated with their affinities towards the tiflucarbine binding site on calmodulin. No such correlation was evident when structurally unrelated reference compounds were tested. The association of tiflucarbine with calmodulin thus appears pharmacologically specific and selective and possibly contributes to the potent antidepressant activity of the drug.

3',5'-Cyclic-AMP Phosphodiesterases↗