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)

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE-cellulose column and assayed for protein phosphatases for tau factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form 2 (minor), were separated. Each peak was in a single form on Sephacryl S-300 column chromatography. Both peaks 1 and 2 dephosphorylated tau factor phosphorylated by Ca2+/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The Km values were in the range of 0.42-0.84 microM for tau factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+ and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40-60 microM. These results suggest that dephosphorylation of tau factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of tau factor.
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PMID:Dephosphorylation of tau factor by protein phosphatase 2A in synaptosomal cytosol fractions, and inhibition by aluminum. 216 75

Protein phosphatases associated with the particulate fraction from rat liver were studied by chromatographing the fraction on a DEAE-cellulose column and assaying the eluate with phosphorylase alpha and glycogen synthase D as substrates. Phosphorylase phosphatase activity emerged as two peaks, termed P-1 and P-2 in order of elution, both of which were inhibited by Mn2+ and Mg2+. P-1 and P-2 were Mr = 50,000 and 32,000 proteins, respectively, and when treated with trypsin, P-1 converted to a form indistinguishable from P-2, to which protein phosphatase inhibitor-2 was a potent inhibitor. Thus P-2 appears to be the catalytic subunit of type-1 protein phosphatase even though it has been degrated proteolytically as evidenced by its relatively low Mr. The elution profile of glycogen synthase phosphatase activity was entirely different. The activity obtained with 5 mM Mn2+ resolved into three peaks, the second-migrating M-2 being the largest. M-2 is an Mr = 70,000 protein; but an attempt to purify it has been unsuccessful giving a product of Mr = 40,000 and closely similar to the type-1 catalytic subunit in properties including inhibition by inhibitor-2. These results suggest that phosphatases P-1 and M-2 have a common catalytic subunit (type-1), which is bound to different "regulatory" subunits. M-2 distributes in glycogen particles and microsomes evenly while P-1 is almost exclusively in microsomes.
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PMID:Characterization of protein phosphatases associated with the particulate fraction from rat liver. 216 61

Ca2(+)-dependent protein phosphatase was purified from scallop adductor smooth muscle by a combination of DEAE-Toyoperal 650S ion exchange chromatographies and gel filtration on Sephacryl S-300. The phosphatase consisted of two subunits having molecular weights of 60 and 19 kDa. Phosphorylated regulatory light chain-a (RLC-a) was dephosphorylated by this phosphatase both in free and bound states in myosin prepared from the opaque portion of scallop smooth muscle (opaque myosin). The dephosphorylation was activated by Ca2+. The half maximal activation was a 1 microM free Ca2+ in the presence of calmodulin and 7 microM free Ca2+ in the absence of calmodulin. Opaque myosin phosphorylated at the heavy chain was not dephosphorylated with this phosphatase. p-Nitrophenyl phosphate was dephosphorylated. In addition to Ca2+, the phosphatase activity for RLC-a was activated by Mn2+, while p-nitrophenylphosphatase activity was activated by Mg2+ more strongly than by Mn2+. The pH-activity curves showed a maximum at pH 7 in the presence of Mn2+, but at around pH 8 in the presence of Mg2+. This phosphatase is similar to phosphatase 2B or calcineurin. The possible regulatory function of this phosphatase in scallop catch muscle is discussed.
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PMID:Ca2(+)-dependent protein phosphatase which dephosphorylates regulatory light chain-a in scallop smooth muscle myosin. 216 91

1. Although Mn2+ could mimic kinase FA/ATP.Mg to activate ATP.Mg-dependent protein phosphatase, strong indications have been obtained that the Mn2(+)-activated and FA/ATP.Mg-activated phosphatase forms are not identical in terms of their substrate specificities and catalytic properties. 2. Both Mn2(+)-activated and FA/ATP.Mg-activated phosphatase forms readily dephosphorylate 32P-labeled phosphorylase a and myelin basic protein (MBP), however the Mn2(+)-activated phosphatase displays activity preferentially against [32P]MBP and FA/ATP.Mg-activated phosphatase preferentially dephosphorylates [32P]phosphorylase a, representing a unique control mechanism to regulate the substrate specificity of multisubstrate protein phosphatase in mammalian tissues.
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PMID:Selective activation of the two catalytic sites in the ATP.Mg-dependent phosphoprotein phosphatase by kinase Fa and Mn2+ ion. 216 37

One p-nitrophenyl phosphate phosphatase (A) and five protein phosphatases (B, C, D, E, F) with neutral pH optimum (7.0-7.5) were partially purified from human platelets. Protein phosphatases were activated by Mn2+ (B-F), Mg2+ (D, F) or Ca2+ (F) but all of them had substantial activity even in the presence of EDTA. The activity of phosphatase D was predominant when assayed in the presence of EDTA. Phosphatase F was significantly enhanced by Ca2+ and calmodulin and therefore considered to be calcineurin. Without strict substrate specificity, all protein phosphatases (B-F) dephosphorylated phosphoproteins like actin binding protein, 47k protein and myosin light chain. Thus, it was suggested that protein phosphatases might play a role in the down regulation of platelet function not only in the resting but agonist-stimulated platelets.
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PMID:Platelet protein phosphatases and their endogenous substrates. 217 85

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.
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PMID:Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone. 241 35

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

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

Purified calcineurin phosphatase is converted upon incubation in millimolar Ni2+ or Mn2+ to an active form by association with these metal activators. The bound metal ion is not dissociable from calcineurin by dialysis or gel filtration, but can be released upon prolonged incubation of the enzyme with Ca2+/calmodulin or chelating agents (Pallen, C.J., and Wang, J.H. (1986) J. Biol. Chem. 261, 16115-16120). The present study has been undertaken to test the possibility that calcineurin in brain may contain tightly bound Ni2+ or Mn2+. A monoclonal antibody (VA1) immunoaffinity matrix was prepared and shown to affect specific precipitation of calcineurin from crude bovine brain extract. Using [3H]-, [63Ni2+]-, and [54Mn2+]calcineurin added to the extract as radioactive tracer, it was found that up to 80% of the calcineurin could be immunoprecipitated, and that more than 50% of the originally bound metal ions could be detected in the immunoprecipitate. When samples of calcineurin immunoprecipitated from brain extracts were analyzed by atomic absorption spectroscopy, Ni2+ and Mn2+ were not detected, whereas, Zn2+, a constitutive metal of calcineurin (King, M. M., and Huang, C. Y. (1984) J. Biol. Chem. 259, 8847-8856) was found in the expected amount. The result suggests that calcineurin in brain does not contain tightly associated Ni2+ or Mn2+.
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PMID:Calcineurin immunoprecipitated from bovine brain extract contains no detectable Ni2+ or Mn2+. 253 14

Binding of epidermal growth factor (EGF) stimulates tyrosyl protein kinase activity of its receptor in the epidermis. This tyrosine residue phosphorylation is thought to be one mechanism by which EGF mediates its effects such as growth stimulation. To modulate a cellular response to EGF, an enzyme which dephosphorylates phosphotyrosyl residues should be present to oppose the effect of the tyrosyl kinase activity of the EGF receptor. We have identified an enzyme in the neonatal mouse epidermis which has the ability to dephosphorylate tyrosyl residues in vitro on EGF receptors. This phosphatase is a soluble protein with a molecular weight greater than 10,000 daltons and shows optimum activity at neutral pH. This epidermal tyrosyl protein phosphatase is not inhibited by tartrate, ATP, and micromolar levels of zinc, but is inhibited by millimolar levels of zinc, magnesium, manganese, and fluoride. Unlike other well-known phosphotyrosyl phosphatases, alkaline phosphatase, and calcineurin, this enzyme is not inhibited by EDTA. Thus, we have identified and partially characterized a possibly unique phosphotyrosyl phosphatase from the epidermis.
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PMID:Identification of a phosphotyrosyl-protein phosphatase in mouse epidermis. 253 66


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