Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The calmodulin contents of rabbit brain, lung, kidney and liver, of bovine aorta and uterus, and of chicken gizzard have been determined. 2. The calmodulin in all of these tissues has been shown to be present in the form of very stable complexes with several other proteins. 3. A calmodulin-binding protein of mol.wt. 22 000 has been purified in high yield from bovine brain. It has been shown to interact with calmodulin and rabbit skeletal-muscle troponin C in a Ca2+-dependent manner. 4. The 22 000-mol.wt. protein inhibits the activation of bovine brain phosphodiesterase by calmodulin, but has very little affect on the activation of myosin light-chain kinase. 5. Calmodulin-binding proteins of mol.wts. 140000, 77000 and 61000 have also been partially purified from rabbit brain by affinity chromatography and have been shown to interact in a Ca2+-dependent manner with calmodulin. 6. The apparent molecular weights of the calmodulin-calmodulin-binding protein complexes, determined by gel filtration in the presence of 6M-urea, have been shown to be similar for most of the mammalian tissues examined. 7. By using 125I-labelled calmodulin, similar complexes have been demonstrated in rabbit skeletal muscle, although they are present at much lower concentrations.
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PMID:Calmodulin-binding proteins from brain and other tissues. 53 97

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.
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PMID:Identification of calmodulin-binding proteins. 238 84

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.
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PMID:Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). 284 52

A heat-stable 32K calmodulin-binding protein has been purified approximately 3,670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin-Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62 mg of pure heat-stable calmodulin-binding protein was obtained from 390 g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (f/fo = 1.89) and consisted of a single polypeptide of Mr = 32,000. It is a highly acidic protein (pI = 3.9) with a diffusion coefficient of 5.4 X 10(-7) cm2/s, a sedimentation coefficient of 1.43 S, and a Stokes radius of 39.5 A in its free form and 41.3 A in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulin-binding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistinguishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011-9016).
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PMID:Establishment of an efficient purification method and further characterization of 32K calmodulin-binding protein in testis. 300 46

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.
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PMID:Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. 302 8

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.
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PMID:The binding of calmodulin to myelin basic protein and histone H2B. 616 7

1. Calmodulin-like proteins were purified from the fruiting bodies of higher (basidiomycete) fungi and barley (Hordeum sp.) shoots. 2. These calmodulins have electrophoretic mobilities on 10% (w/v) polyacrylamide gels at pH 8.3 in the presence of 6 M-urea and at pH 8.3 in the presence of 0.1% sodium dodecyl sulphate similar to that of bovine brain calmodulin. They interacted with rabbit skeletal-muscle troponin I in the presence of Ca2+. 3. Barley and fungal calmodulins activated myosin light-chain kinase and phosphodiesterase in the presence of Ca2+, although the amounts needed were at least an order of magnitude greater than is required to produce the same effect with mammalian calmodulin. 4. Amino acid analyses indicated a number of differences from the mammalian protein, most notably the absence of trimethyl-lysine. 5. By using 125I-labelled calmodulin, a small amount of calmodulin-binding protein was detected in homogenates of barley and fungi. 6. No protein corresponding to calmodulin could be found in Escherichia coli or yeast, although a relatively high concentration of a protein that bound calmodulin was detected in E. coli by this technique.
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PMID:The preparation of calmodulins from barley (Hordeum sp.) and basidiomycete fungi. 624 33

Calmodulin is a ubiquitous, multifunctional, Ca2+-dependent regulatory protein, controlling a wide variety of Ca2+-mediated reactions. The versatility of calmodulin raises the question of how it exerts specificity at the molecular level. Cyclic nucleotide phosphodiesterase consists of multiple forms, one of which requires calmodulin for full activity. Calcineurin, a calmodulin-binding protein, inhibits the calmodulin-stimulated phosphodiesterase activity by competing with the enzyme for calmodulin. In this report, we present experiments which indicate that, although calcineurin potentially inhibits calmodulin-supported enzyme activity, its effectiveness as an inhibitor depends on the level of cAMP. In the presence of elevated levels of cAMP, the affinity of calmodulin for phosphodiesterase increased markedly, but that for calcineurin was not altered. Thus, the enzyme became relatively refractory to inhibition by calcineurin. This finding suggests that an increase of cellular cAMP could lead to a condition favorable to its own hydrolysis and that this phenomenon might represent an example of molecular specificity in calmodulin-regulated reactions.
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PMID:cAMP renders Ca2+-dependent phosphodiesterase refractory to inhibition by a calmodulin-binding protein (calcineurin). 626 Jul 98

An inhibitory factor for Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase of bovine brain was present in the soluble fraction of Escherichia coli. The factor was heat-stable but trypsin sensitive. The activity of brain phosphodiesterase supported by Ca2+ and calmodulin was inhibited by the factor in a dose dependent manner, but the basal activity was not affected. The inhibition of phosphodiesterase induced by the factor could be abolished by adding large amount of calmodulin, but not by increasing concentration of Ca2+. It was suggested that the factor interacted with calmodulin and thereby inhibited the phosphodiesterase. The factor may be a calmodulin-binding protein in E. coli.
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PMID:Inhibition of bovine brain cyclic nucleotide phosphodiesterase by a proteinaceous factor from Escherichia coli. 628 29

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein.
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PMID:Characterization of a calmodulin-binding protein that is deficient in trifluoperazine-resistant variants of the macrophage-like cell line J774. 657 95


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