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)

Calcineurin is one of the calmodulin binding proteins and a Ca2+-dependent and calmodulin-stimulated phosphoprotein phosphatase. We used antisera to the calcineurin as a cell-type-specific marker in order to identify neuronal cells in the rat brain and human neoplasms. In normal rat brain slices, basal ganglia were stained macroscopically, and other areas such as cerebral cortex, corpus callosum, cerebellar cortex, granular layer and pyramidal tract of the spinal cord were lightly identified as well. Under the light microscope, it was found that only the neuronal cells were stained, and astrocytes, oligodendrocytes, ependymal cells and vessels were not. Intracellular distribution of the staining showed various patterns and staining intensity of varying degree. Using the PAP method, localization of the calcineurin in formalin-fixed, paraffin-embedded tissues were studied in 65 human intracranial neoplasms, and in 11 human extracranial neoplasms. The neuronal elements of neuroblastoma, ganglioglioma, ganglioneuroma and retinoblastoma were clearly stained. In contrast, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, meningioma, neurinoma, pituitary adenoma, craniopharyngioma, hemangioblastoma, hamartoma, lymphoma and mesenchymal tumor were all negative. Two cases out of 5 medulloblastomas were stained, but others were not. Although positive tumors disclosed various staining patterns and intensities, these results indicated that calcineurin could be a new neuronal marker in human brain tumors.
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PMID:Calcineurin as a neuronal marker of human brain tumors. 242 51

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.
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PMID:Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase. 242 55

'Wash-out' and inactivation of the Ca current were examined in dialysed, voltage-clamped neurones of Helix aspersa under conditions that isolate the Ca current virtually free of other currents. EGTA or other internal Ca2+ chelators were routinely omitted from the dialysate. The time-dependent loss, or wash-out, of Ca current was slowed by addition to the dialysing solution of agents, such as dibutyryl adenosine 3'-5'-cyclic monophosphate (dibutyryl cyclic AMP), Mg adenosine 5'-triphosphate (ATP) and the catalytic subunit of cyclic-AMP-dependent protein kinase, that promote protein phosphorylation and by EGTA. However, neither the phosphorylation-promoting agents nor internal EGTA prevented wash-out entirely, nor did they significantly restore previously 'washed-out' current. With phosphorylating agents in the dialysing solution, the irreversible development of wash-out was greatly reduced by introduction of leupeptin, an inhibitor of protease activity. Thus, the irreversible component of wash-out appears to result from a Ca-dependent proteolytic process. In the presence of leupeptin alone, Ca current amplitude continued to decline: however, the current could be largely or fully restored with addition of catalytic subunit, dibutyryl cyclic AMP, and Mg ATP to the dialysing solution. Thus, inhibition of proteolysis revealed a reversible component of wash-out that appears to result from dephosphorylation. During perfusion with leupeptin, Mg ATP, dibutyryl cyclic AMP and catalytic subunit the Ca current remained stable for up to several hours without addition of internal Ca2+ buffer. The rate of inactivation of the current that occurs during a depolarizing step showed only a very gradual decline during this time. Under these conditions, perfusion with calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation. This inactivation was virtually eliminated by introduction of EGTA or by replacement of external Ca2+ with Ba2+, which is consistent with the ion dependency for calmodulin-dependent activation of calcineurin. When ATP in the dialysate was replaced with ATP-gamma-S (adenosine 5'-O-(thiotriphosphate], an analogue that donates a thiophosphate group resistant to hydrolysis, the rate of inactivation slowed. Since Ca-dependent inactivation during step depolarizations is enhanced by conditions that promote dephosphorylation, and Ca current wash-out is slowed by conditions that promote phosphorylation, inactivation and reversible wash-out appear to be related.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. 243 51

Myelin basic protein (MBP) reduces the amount of phosphatase activity produced in the kinase FA-mediated activation of the ATP,Mg-dependent phosphatase. MBP was shown not only to inhibit the activated enzyme, but also to impair the kinase FA-mediated activation of the inactive phosphatase. In addition MBP prevents the time-dependent inactivation of the catalytic subunit by the modulator protein. These observations point to a regulatory role for MBP in the reversible activation of the ATP,Mg-dependent protein phosphatase by kinase FA.
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PMID:Interaction of myelin basic protein with the different components of the ATP,Mg-dependent protein phosphatase system. 243 57

Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enzymatic regulation of calcium current in dialyzed and intact molluscan neurons. 243 95

Calcineurin, a multifunctional Ca2+ (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase, has been reported to be present in the striatal neurons which project to the globus pallidus and the substantia nigra. In the present study, we examined what types of cells in the rat striatum express calcineurin. The calcineurin-positive neurons were of medium size (mean diameter of 16 microns) and constituted about 60-70% of the total neuronal population in the striatum. Under light microscopy, the calcineurin-positive neurons had round, triangular, or polygonal cell bodies with a relatively small amount of cytoplasm. Electron microscopic examination of 20 randomly selected striatal calcineurin-immunoreactive neurons revealed that their nuclei did not show any invaginations or intranuclear inclusions. The calcineurin-positive neurons were characterized by Golgi impregnation as the densely spinous type. On the other hand, it was demonstrated that calcineurin-positive neurons are a separate population from the diisopropylfluorophosphate-acetylcholinesterase-positive cells or nicotinamide adenine dinucleotide phosphate diaphorase-positive cells, by means of the combination of immunocytochemistry and enzyme histochemistry. In addition, simultaneous localization of calcineurin and substance P in a single cell was observed in some striatal neurons using a double immunostaining method. On the basis of these findings, it was considered that most calcineurin-immunoreactive neurons in the rat striatum may be classified as medium-size densely spiny neurons.
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PMID:Morphological characterization of the rat striatal neurons expressing calcineurin immunoreactivity. 244 61

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.
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PMID:Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase. 244 76

Three monoclonal antibodies (MAb's) against bovine brain calcineurin, a Ca++-calmodulin dependent phosphatase, have been developed and characterized. Among these antibodies, two are alpha-subunit (60,000 Mr) specific and one is beta-subunit (19,000 Mr) specific. These antibodies also cross-react with similar proteins obtained from brains of human, rat and mouse. The methodology for raising these antibodies and their properties are described.
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PMID:Development and characterization of monoclonal antibodies to bovine brain calcineurin. 244 54

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).
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PMID:Characterization of a high-affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A. 247 57

In vitro phosphorylation of purified human plasma apolipoprotein A-I (apoA-I) by a recently characterized Ca2+/calmodulin-dependent kinase (Beg, Z. H., Stonik, J. A., and Brewer, H. B., Jr. (1987) J. Biol. Chem. 262, 13228-13240) was time-, Ca2+-, and calmodulin-dependent. Maximal phosphorylation of human apoA-I revealed a stoichiometry of approximately 1 mol of PO4/mol of apoA-I. Phosphorylation of apoA-I resulted in an increase of two negative charges and consequently a shift to a more acidic pI for each apoA-I isoform following isoelectrofocusing. Dephosphorylation of 32P-apoA-I with either phosphatase I or a Ca2+/calmodulin-dependent phosphatase was associated with a virtually complete loss of 1 mol of 32PO4/mol of apoA-I. Phosphoamino acid analysis of a purified 32P-peptide established that the phosphorylation occurred on a single serine residue. Automated Edman degradation of the purified 32P-peptide revealed a single amino acid sequence and indicated that phosphorylation occurred on the serine at residue 201 in the apoA-I sequence. ApoA-I was shown to be secreted as a phosphoapolipoprotein by HepG-2 cells as well as primary human hepatocytes. Analysis of HepG-2 cells established that intracellular apoA-I, like secreted apoA-I, is phosphorylated. Dephosphorylation of both secreted and intracellular 32P-apoA-I revealed the loss of radioactivity in the apoA-I protein bands. These data provide the initial description of a post-translational modification involving reversible phosphorylation of extracellular as well as intracellular apoA-I on a serine residue. These combined results suggest that synthesis and secretion of apoA-I as a phosphoapolipoprotein in HepG-2 cells as well as primary human hepatocytes may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion.
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PMID:Human apolipoprotein A-I. Post-translational modification by covalent phosphorylation. 249 23


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