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PubMed1992

Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. An alpha-factor inducible gene.

A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes. The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.

Amino Acid Sequence↗
PubMed1997

Characterization of mutant Bordetella pertussis adenylate cyclase toxins with reduced affinity for calmodulin. Implications for the mechanism of toxin entry into target cells.

Bordetella pertussis secretes a calmodulin-stimulated adenylate cyclase toxin (CyaA) that is one of the major virulence factors of this organism. The toxin is able to enter various types of eukaryotic cells where, upon activation by calmodulin, it catalyzes the production of non-physiological amounts of cyclic AMP. The mechanism of toxin entry into target cells is unknown, although it has been shown that it does not involve receptor-mediated endocytosis. The adenylate cyclase toxin exhibits a very high affinity for calmodulin, and it has been proposed that the energy of calmodulin-binding to CyaA might be required for the entry of the toxin into the target cells [Oldenburg, D.J., Gross, M. K., Wong, C. S. & Storm, D. R. (1992) Biochemistry 31, 8884-8891]. In the present study, we have reexamined this issue by analyzing the cytotoxicity of various modified CyaA toxins that have altered calmodulin affinity. We show that despite their low affinity for calmodulin (at least 1000-times less than that of the wild type CyaA), these toxins were able to efficiently deliver their catalytic domain into the cytoplasm of the target cells, erythrocytes. These results demonstrate that high-affinity calmodulin binding is not required for the entry of B. pertussis adenylate cyclase into eukaryotic cells. However, the high-affinity of CyaA for calmodulin is crucial for an efficient synthesis of cAMP within the target cells.

Adenylyl Cyclases↗
PubMed1988

Distribution of calmodulin- and cyclic AMP-stimulated protein kinases in synaptosomes.

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.

Adenosine Triphosphate↗
PubMed1987

Effects of lithium on calmodulin-stimulated adenylate cyclase activity in cortical membranes from rat brain.

The influence of lithium on calmodulin-stimulated adenylate cyclase activity has been studied in vitro and after chronic treatment. Chronic lithium treatment decreased calcium-calmodulin-stimulated adenylate cyclase activity in rat cortical membranes, while no effect was observed on GTP-stimulated activity. Lithium in vitro inhibited adenylate cyclase activity stimulated by isoprenaline, GTP or calcium-calmodulin. Calcium-calmodulin-stimulated activity was more sensitive to lithium (2 mM) than isoprenaline- and GTP-stimulated activities (5 mM) and activities by these agents combined. Lithium had no effect on the unstimulated enzyme activity. The inhibitory effect of lithium in vitro on calcium-calmodulin-stimulated adenylate cyclase activity was antagonized by magnesium. The inhibition induced by lithium in vitro on the GTP-stimulated adenylate cyclase activity was increased by substituting manganese for magnesium in the assay media. Furthermore, the manganese-stimulated activity was also reduced by lithium. The latter effect was not observed in calmodulin-depleted membranes, but the inhibitory effect of lithium could be restored by addition of exogenous calmodulin. The present results suggest that lithium might influence the interaction of calmodulin with the enzyme and/or interfere with the divalent cation site(s) on the adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1995

Enhancing effect of calmodulin on Ca(2+)-induced Ca2+ release in the sarcoplasmic reticulum of rabbit skeletal muscle fibres.

1. We analysed the effect of calmodulin on Ca(2+)-induced Ca2+ release (CICR) in the sarcoplasmic reticulum (SR) using chemically skinned fibres of rabbit psoas muscle. Ca2+ release was measured using fura-2 microfluorometry. 2. In saponin-skinned fibres, calmodulin potentiated Ca2+ release at low Ca2+ concentrations (< 3 microM), while it showed an inhibitory effect at high Ca2+ concentrations (3-30 microM). 3. Co-application of ryanodine and calmodulin at 0.3 microM Ca2+, but not ryanodine alone, induced a decline in the Ca2+ uptake capacity of the SR, an effect expected from the open-lock of active CICR channels by ryanodine. Thus, potentiation of Ca2+ release by calmodulin at low Ca2+ concentrations can be regarded as a result of the activation of the ryanodine receptor. 4. Greater concentrations of calmodulin were required for potentiation of CICR at low Ca2+ concentrations (1 microM) than for inhibition at high Ca2+ concentrations (10 microM). 5. In beta-escin-permeabilized fibres in which intrinsic calmodulin was retained, the rates of CICR were similar to those measured in the presence of 1 microM calmodulin in saponin-permeabilized fibres. 6. These results suggest that calmodulin plays an important role in the regulation of CICR channels in intact skeletal muscle fibres.

Animals↗
PubMed1993

Dependence of calmodulin localization in the retina on the NINAC unconventional myosin.

Calmodulin is a highly conserved regulatory protein found in all eukaryotic organisms which mediates a variety of calcium ion-dependent signalling pathways. In the Drosophila retina, calmodulin was concentrated in the photoreceptor cell microvillar structure, the rhabdomere, and was found in lower amounts in the sub-rhabdomeral cytoplasm. This calmodulin localization was dependent on the NINAC (neither inactivation nor afterpotential C) unconventional myosins. Mutant flies lacking the rhabdomere-specific p174 NINAC protein did not concentrate calmodulin in the rhabdomere, whereas flies lacking the sub-rhabdomeral p132 isoform had no detectable cytoplasmic calmodulin. Furthermore, a defect in vision resulted when calmodulin was not concentrated in the rhabdomeres, suggesting a role for calmodulin in the regulation of fly phototransduction. A general function of unconventional myosins may be to control the subcellular distribution of calmodulin.

Animals↗
PubMed1991

Interspecies comparison of calmodulin binding proteins throughout the gastrointestinal tract: comparison with human colon adenomas and adenocarcinomas.

Calmodulin is an ubiquitous cytoplasmic protein which mediates many of the actions of calcium on intestinal tissue including regulation of growth and differentiation of normal and neoplastic cells. Using a biotinylated calmodulin overlay system, we compared the pattern of calmodulin binding proteins throughout the gastrointestinal tract of mice, rats, rabbits, and humans, and in human colonic adenomas and adenocarcinomas. A common calmodulin binding protein of 67 kDa was found in membrane and cytosolic fractions of oesophagus, stomach, proximal and distal small intestine, and colon from all four species. In human tissue this 67 kDa protein was present in greatest concentration in stomach tissue. Furthermore, a 67 kDa binding protein was the major calmodulin binding protein from human stomach and ileum as determined by ion exchange and calmodulin affinity chromatography. A similar pattern of binding proteins was noted between rabbit and human cytosolic fractions; proteins of 60/67 kDa and 105 kDa were present in stomach tissue. A 94 kDa protein was present in samples of rabbit and human ileum but not of mouse or rat. A similar pattern of calmodulin binding proteins was seen in normal and neoplastic large bowel tissue, apart from one of nine adenocarcinomas, where a distinct 54 kDa band was noted in both cytosolic and membrane fractions. The results of this study show interspecies and organ differences between calmodulin binding proteins, but suggest that a 67 kDa protein is the major binding protein present throughout normal gastro-intestinal tract and neoplastic human tissue.

Adenocarcinoma↗
PubMed1996

Calmodulin glycation in diabetic rat lenses.

The activation of phosphodiesterase by calmodulin isolated from diabetic rat lens tissue was determined. Male rats, 200-250 g, were rendered diabetic by injection of streptozotocin (45 mg/kg body weight) via the tail vein. After the onset of diabetes, the animals were observed for 15 weeks. Compared with a blood glucose level of 94.84 +/- 2.72 mg/dL in the control group, 1, 4, and 6 week diabetic rats had blood glucose levels of 357.00 +/- 7.55, 366.53 +/- 4.76, and 366.57 +/- 5.30 mg/dL, and 8, 10, and 15 week diabetic rats had levels over 400 mg/dL. After collecting the lens tissue, boiled extracts were prepared as a calmodulin source and were applied to a Glycogel B column for separating glycosylated and nonglycosylated calmodulin. Calmodulin activities of boiled extracts and glycosylated eluates were determined via the activation of calmodulin-deficient brain phosphodiesterase. Calmodulin activities of diabetic rat lenses were significantly reduced compared with the control group. The results of glycosylated protein determination with the thiobartiuric acid method were in accordance with calmodulin activities. Calmodulin activities of the diabetic lens tissues were reduced, while the protein glycosylation levels were elevated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Platelet membrane calmodulin-stimulated calcium-adenosine triphosphatase. Altered activity in essential hypertension.

Platelet free Ca2+ concentration has been found to be elevated in essential hypertension and to correlate with blood pressure level. Free cytoplasmic calcium concentration is determined by calcium influx, pooling, and efflux. The present study found a Ca2+-ATPase in platelet membranes that has a high affinity for Ca2+ (Km approximately 1 microM), is inhibited by low concentrations of orthovanadate (Ki approximately 1 microM), and can be stimulated by calmodulin (Km approximately 5 nM). The absolute increase in calmodulin-stimulated Ca2+-ATPase activity was not different between normotensive and hypertensive subjects; however, the degree of stimulation of Ca2+-ATPase activity at saturating calmodulin concentrations apparently was diminished in calmodulin-deficient membranes from subjects with established essential hypertension (40%) as compared to that in normotensive subjects of similar age (135%; p less than 0.001). Affinities for calmodulin and Ca2+ were comparable between the two groups, while the capacity for Ca2+-ATPase activity (basal and calmodulin-stimulated) was markedly greater (1.5- to 1.8-fold) in both native and calmodulin-deficient membranes from hypertensive subjects. On the other hand, the defective calcium efflux pump activity, as assessed by a decreased degree of calmodulin stimulation, may have contributed to elevated cytoplasmic calcium concentrations and the associated enhanced hormone sensitivity in platelets from essential hypertensive subjects. This may represent an adaptive negative feedback control mechanism to protect the cell against Ca2+ overload.

Adult↗
PubMed1989

Calmodulin modulation of single sarcoplasmic reticulum Ca2+-release channels from cardiac and skeletal muscle.

Sarcoplasmic reticulum (SR) contains a Ca2+-conducting channel that is believed to play a central role in excitation-contraction coupling by releasing the Ca2+ necessary for muscle contraction. The effects of calmodulin on single cardiac and skeletal muscle SR Ca2+-release channels were studied using the planar lipid bilayer-vesicle fusion technique. Calmodulin inhibited Ca2+-release channel opening by reducing the mean duration of single-channel open events without having an effect on single-channel conductance. Inhibition by calmodulin was dependent on Ca2+ concentration and occurred in the absence of ATP. The effects of calmodulin were reversed by mastoparan, a calmodulin-binding peptide. Two other calmodulin antagonists [calmidazolium and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide] modified the gating behavior of the channel in the absence of exogenous calmodulin in a concentration- and Ca2+-dependent manner. Our results suggest that calmodulin can modulate excitation-contraction coupling by directly interacting with the SR Ca2+-release channel of cardiac and skeletal muscle.

Animals↗
PubMed2000

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

Animals↗
PubMed1990

Calcium-calmodulin binding in ischemic rat neurons after calcium channel blocker therapy.

Calcium channel blockers such as nicardipine improve outcome after global cerebral ischemia and may attenuate ischemic neuronal injury by preventing calcium influx and binding to calmodulin. We followed the temporal and regional sequence of neuronal calcium-calmodulin binding in normal rats (n = 6), untreated ischemic rats (n = 15), and ischemic rats treated with 0.05 mg/kg/hr s.c. nicardipine (n = 13). After 30 minutes of four-vessel occlusion, 40-microns brain sections were incubated in an anti-calmodulin antibody specific for calmodulin not bound to calcium and brain protein. Light-microscopic sections were examined immediately after ischemia and after 2 and 24 hours of reperfusion. Extensive staining of unbound calmodulin was seen in all hippocampal regions and in the cortex in normal rats. In untreated ischemic control rats, staining was lost, indicating calcium-calmodulin binding immediately after ischemia in all regions. However, after 24 hours, staining returned to normal in the cortex and dentate, and minimal staining returned in CA1 and CA3. Nicardipine-treated animals had significantly less calcium-calmodulin binding in CA1 and in the dentate after 2 hours of reperfusion. This study demonstrates that in clinically relevant doses nicardipine has a limited effect on calcium-calmodulin binding in selectively vulnerable regions after severe ischemia.

Animals↗
PubMed1986

Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

Animals↗
PubMed2002

Interaction of calmodulin with the cytoplasmic domain of platelet glycoprotein VI.

The platelet collagen receptor, glycoprotein VI (GPVI), and GPIb-IX-V, which binds von Willebrand factor, initiate platelet aggregation at low or high shear stress, respectively. We recently reported that positively charged, membrane-proximal sequences within cytoplasmic domains of GPIbbeta and GPV of GPIb-IX-V bind calmodulin. We now show that GPVI also binds calmodulin as follows-(1) calmodulin coimmunoprecipitated with GPVI from resting platelet lysates using an anti-GPVI IgG, but partially dissociated in platelets activated by collagen or collagen-related peptide; (2) calmodulin coprecipitated from platelet lysates with maltose-binding protein (MBP)-GPVI cytoplasmic domain fusion protein, but not MBP alone; (3) GPVI-related synthetic peptide based on the membrane-proximal sequence, His269-Pro287, induced a shift in calmodulin migration on nondenaturing gels, an assay that identifies calmodulin-binding peptides. His269-Pro287 is analogous to the calmodulin-binding sequence in GPIbbeta. The novel interaction of GPVI and calmodulin may regulate aspects of GPVI function.

Amino Acid Sequence↗
PubMed1985

The effect of thyroid status on the calmodulin content of several tissues in the rat.

The present studies represent an initial effort to evaluate a possible relationship between thyroid status and calmodulin and were undertaken because of evidence that some actions of the thyroid hormones are calmodulin mediated. To this end, the effects of hypothyroidism and of thyroid hormone excess on the concentration of calmodulin in several tissues of the rat were evaluated. In thymocytes, liver, heart, and brain, but not in skeletal muscle, calmodulin concentrations were markedly increased in the thyrotoxic rat (140-203% of values in normal controls); values were unchanged in hypothyroid animals. These changes were evident regardless of whether calmodulin concentrations were expressed per cell (thymocytes), or in relation to either the wet wt or protein content of the tissues. These findings are apparently the first to demonstrate that thyroid hormone, unlike other hormones tested thus far, can influence tissue calmodulin content. In view of the fact that many metabolic systems that are influenced by calmodulin are similarly affected by thyroid hormones, the findings support the possibility that some actions of thyroid hormone are calmodulin mediated.

Animals↗
PubMed1989

Analysis of expression of multiple genes encoding calmodulin during spermatogenesis.

It has recently been determined that the intracellular calcium receptor, calmodulin, is encoded by a multigene family. At least three calmodulin genes that encode the identical protein are expressed in adult testis from a variety of mammalian species. The complementary techniques of RNA blot hybridization and in situ hybridization were used to assess the relative levels of calmodulin RNAs during rat testis cell differentiation. RNA isolated from highly purified populations of somatic or germinal cells and sections of fixed testis were analyzed for hybridization to specific probes corresponding to the three separate rat calmodulin genes. The level of each calmodulin RNA can be described by a unique developmental pattern during spermatogenesis. The steady state levels of transcripts corresponding to calmodulin gene I (CaM I) and III increase in early meiotic cells (leptotene-zygotene spermatocytes) and remain constant throughout meiosis. The level of the CaM II RNA increases between early and mid-pachytene spermatocytes, but is only transiently elevated. The level of this RNA decreases in round spermatids and is almost nondetectable in cytoplasts shed from elongating spermatids. The levels of CaM I RNAs are maintained in early round spermatids, but only RNA derived from the CaM III gene is still evident in late spermatids. The data indicate that an individual germ cell contains RNAs derived from multiple calmodulin genes and suggests that the divergent calmodulin genes may respond to different cellular regulatory signals.

Animals↗
PubMed1980

Purification and characterization of calmodulin from sea urchin spermatozoa.

Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85 degrees C, ammonium sulphate precipitation at pH 4.2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8.3 micrograms calmodulin were recovered per 10(10) sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.

Amino Acids↗
PubMed1983

The relationship between prolactin secretion and calmodulin activity.

The possible role of calmodulin in the control of prolactin secretion was examined. The effects of a wide variety of agents on inhibition of prolactin secretion from isolated rat anterior pituitary cells and inhibition of calmodulin activity in an in-vitro system (calmodulin-activated cyclic GMP phosphodiesterase) were compared. A number of phenothiazines showed a close correlation of potencies in the two systems, as did the more specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W7), suggesting that calmodulin may be involved in the control of prolactin secretion. Some other drugs also inhibited both prolactin secretion and calmodulin activity in addition to their other well-characterized biochemical effects. However, many of these drugs (including two phenothiazines) were more potent inhibitors of prolactin secretion than of calmodulin activity, suggesting that other intracellular systems in addition to calmodulin may be involved in the control of the secretory process.

Animals↗