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 distribution of phosphoprotein phosphatase (PPPase) and estrogen-induced protein (IP) from 19- to 20-day-old rat uteri before and after fractionation of uterine cytosol, by ammonium sulfate, and by preparative cellulose acetate gel electrophoresis was studied. There was a lack of significant difference between the specific activity of PPPase or its electrophoretic mobility in control extracts and those of extracts made 1 hour after injection of the rats with 5 mcg estradiol-17beta. Most of the recovered PPPase activity appeared in the fraction precipitating between 0-50% saturation with ammonium sulfate. Most of the IP is found at 50-80% saturation, and less than 10% of the PPPase activity. A single peak of PPPase activity was shown at a mobility of .5 relative to bovine serum albumin with electrophoresis of the 50-80% ammonium sulfate fraction. A peak of IP with mobility of 1.2 was also shown. ''The results are incompatible with the view that IP mediates estrogen action by virtue of its PPPase activity.''
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PMID:Proceedings: Separation of the "estrogen-induced protein" from phosphoprotein phosphatase activity of immature rat uteri. 17 73

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.
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PMID:Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12. 138 93

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.
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PMID:Immobilized inhibitor-1 binds and inhibits protein phosphatase 1. 254 51

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.
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PMID:Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II. 254 44

A polypeptide from a tryptic digest of bovine serum albumin potentiates glucose oxidation stimulated by insulin in isolated rat adipocytes. We studied whether this effect is related to a modification of the insulin receptor kinase. In a solubilized rat adipocytes receptor system, the peptide caused dose-dependent inhibition of the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of insulin receptor. The peptide also inhibited stimulation by vanadate of tyrosine autophosphorylation of the beta subunit of the receptor, though it enhanced vanadate-stimulated glucose oxidation. During the phosphorylation reaction, no phosphorylated forms of the peptide could be detected. The peptide had no effect on dephosphorylation of the phosphorylated beta subunit of the insulin receptor. These results strongly suggest that the inhibition of phosphorylation by the peptide is due not to either simple substrate competition or activation of phosphoprotein phosphatase, but to specific inhibition of tyrosine-specific protein kinase.
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PMID:Inhibition of tyrosine autophosphorylation of the solubilized insulin receptor by an insulin-stimulating peptide derived from bovine serum albumin. 355 83

Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.
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PMID:Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose. 608 42

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.
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PMID:Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions. 625 90

In this report we describe a novel in vitro phenomenon involving the interaction of insulin with purified protein phosphatases. Evidence is presented that porcine insulin is capable of activating and binding to rabbit skeletal muscle protein phosphatases in vitro. Its effects were examined on four rabbit skeletal muscle protein phosphatases. Two of these, phosphatases C-I and C-II, are of Mr approximately 35,000 and are the dissociated forms of protein phosphatase. The two other phosphatases, H-I and H-II, have Mr approximately 250,000 by gel filtration and represent nondissociated forms of phosphatase. Insulin reproducibly activated homogeneous preparations of protein phosphatase C-II and H-II approximately 3-5-fold in vitro. The activation was dependent on temperature, time, and insulin concentration. The activities of the phosphatases toward both phosphorylase alpha and histone were affected, indicating that this was not a substrate-directed effect. The activation phenomenon was not mimicked by insulin A or B chains, somatostatin, glucagon, or bovine serum albumin, and could be prevented by insulin antiserum. 125I-Insulin was shown to bind to the protein phosphatases by solid phase binding assays. Phosphatases C-I, C-II, and H-II, but not phosphatase H-I, were found to bind insulin reversibly. Half-maximal binding to the protein phosphatases was observed at approximately 5 X 10(-10) M insulin. Labeled insulin was found to coelute with protein phosphatase H-II on gel filtration when a mixture of the two was chromatographed, providing evidence for the formation of an enzyme-insulin complex. These findings suggest that certain protein phosphatases may have a specific binding site(s) for insulin and that these insulin-phosphatase complexes may also exhibit enhanced catalytic activity.
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PMID:A novel in vitro interaction of insulin with rabbit skeletal muscle protein phosphatases. 632 53

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.
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PMID:Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments. 884 29

As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks. Okadaic acid, a protein phosphatase inhibitor, disrupted the hepatocytic cytokeratin network and inhibited autophagy completely in intact hepatocytes, perhaps suggesting that autophagy might be dependent on intact intermediate filaments. Vinblastine and cytochalasin D, which specifically disrupted microtubules and microfilaments, respectively, had relatively little (25-30%) inhibitory effect on autophagic sequestration. In a cryo-ultrastructural study, the various autophagic-lysosomal vacuoles were immunogold-labelled, using the cytosolic enzyme superoxide dismutase as an autophagic marker, Lgp120 as a lysosomal membrane marker, and bovine serum albumin as an endocytic marker. Vinblastine (50 microM) was found to inhibit both autophagic and endocytic flux into the lysosomes, with a consequent reduction in lysosomal size. Asparagine (20 mM) caused swelling of the lysosomes, probably as a result of the ammonia formation that could be observed at this high asparagine concentration. Autophagosomes and amphisomes (autophagic-endocytic, prelysosomal vacuoles) accumulated in asparagine-treated cells, reflecting an inhibition of autophagic flux that might be a consequence of lysosomal dysfunction.
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PMID:Structural aspects of autophagy. 886 Sep 99


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