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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.
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PMID:Functional significance of the central helix in calmodulin. 284 23

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.
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PMID:Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist. 284 68

Purified bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) contains isozymes that are composed of two distinct subunits with molecular masses of 60,000 and 63,000 daltons. Analysis by NaDodSO4 gel electrophoresis and autoradiography of a phosphodiesterase sample phosphorylated in the presence of [32P]ATP and bovine heart cAMP-dependent protein kinase catalytic subunit revealed that only the 60-kDa subunit was phosphorylated. By using an isozyme preparation greatly enriched with the 60-kDa subunit, the following observations regarding the subunit phosphorylation were made. First, the phosphorylation resulted in the maximal incorporation of about 2 mol of phosphate per mol of subunit. Second, complete inhibition of 60-kDa subunit phosphorylation was approached at a saturating concentration of Ca2+ when a molar ratio of calmodulin to phosphodiesterase of 2:1 was used. No inhibition was observed in the presence of either Ca2+ or calmodulin alone. Third, the phosphorylation was accompanied by a decrease in the enzyme affinity for calmodulin; calmodulin concentrations required for 50% activation of nonphosphorylated and maximally phosphorylated phosphodiesterase isozyme samples were 0.51 and 9.3 nM, respectively. Fourth, the phosphodiesterase isozyme could be dephosphorylated by the calmodulin-dependent phosphatase (calcineurin) in the presence of Ni2+ or Mn2+, the dephosphorylation being associated with an increase in the enzyme affinity for calmodulin. Fifth, peak II rabbit liver phosphoprotein phosphatase catalytic unit did not catalyze the dephosphorylation of the phosphodiesterase isozyme.
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PMID:Differential regulation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes by cyclic AMP-dependent protein kinase and calmodulin-dependent phosphatase. 298 24

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.
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PMID:Selective affinity chromatography with calmodulin fragments coupled to sepharose. 298 37

Occupancy of one of the two phenothiazine-binding sites on calmodulin does not significantly decrease the affinity of calmodulin for its target proteins; however, it does affect the ability of calmodulin to activate some enzymes. Previously we demonstrated that a covalent adduct of calmodulin with one molecule of phenothiazine (CAPP1-calmodulin) is an antagonist for the calmodulin-dependent enzymes, cAMP phosphodiesterase and myosin kinase, and a partial agonist for calcineurin. We now show that CAPP1-calmodulin is a full agonist for glycogen synthase kinase and phosphorylase kinase. Unlike phenothiazines, CAPP1-calmodulin is specific for calmodulin-regulated proteins; it has no effect on protein kinase C. With the exception of phosphorylase kinase, occupancy of two phenothiazine-binding sites completely eliminates the ability of calmodulin to activate these proteins. Thus, the study of the interaction of CAPP1-calmodulin with calmodulin target proteins demonstrates that calmodulin interacts differently with different proteins. This is confirmed by studies of the effect of calmodulin fragments, 1-77 and 78-148, on calmodulin-regulated enzymes.
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PMID:Selective effects of CAPP1-calmodulin on its target proteins. 298 45

The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP-dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8).
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PMID:Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role. 300 Jan 46

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of calcineurin from bovine brain. 300 May 60

A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.
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PMID:Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit. 302 30

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.
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PMID:Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody. 302 48

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.
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PMID:Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons. 302 62


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