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:1.11.1.7 (peroxidase)
65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
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PMID:A microtiter-based assay for the detection of protein tyrosine kinase activity. 170 96

H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.
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PMID:H2O2 has a role in cellular regulation. 207 30

It was established previously that the 15-kDa protein phosphorylated in 3T3-L1 adipocytes treated with insulin and phenylarsine oxide is O-phospho-Tyr19 422(aP2) protein, a fatty acid-binding protein. To assess its capacity to serve as substrate of the insulin receptor tyrosine kinase in vitro, native 422(aP2) protein was isolated from 3T3-L1 adipocytes and purified to homogeneity. Receptor-catalyzed phosphorylation of 422(aP2) protein on Tyr19 was markedly activated when long-chain fatty acid, e.g. oleic acid, is bound to the protein. Fatty acid had no effect on autophosphorylation of the insulin receptor by its intrinsic tyrosine kinase. Both saturated (C14, C16, and C18) and unsaturated (all cis-delta 9 C16, -delta 9 C18, and -delta 9,12 C18, -delta 9,12,15 C18, and -delta 5,8,11,14 C20) fatty acids caused substrate activation. The Km for 422(aP2) protein was greatly reduced (from 170 to 3 microM) by oleic acid with little or no effect on Vmax. Upon binding fatty acid to 422(aP2) protein the susceptibility of Tyr19 and Tyr128 to iodination by the lactoperoxidase method increased greatly. These results indicate that upon binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19, which lies within a consensus-type sequence for tyrosine kinase substrates, becomes accessible for phosphorylation by the insulin receptor tyrosine kinase and to iodination by lactoperoxidase.
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PMID:Insulin receptor tyrosine kinase-catalyzed phosphorylation of 422(aP2) protein. Substrate activation by long-chain fatty acid. 217 34

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.
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PMID:Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype. 271 45

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.
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PMID:Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes. 328 33

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.
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PMID:Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues. 636 23

Growth hormone (GH) produces insulin-like effects in rat adipocytes that have been deprived of GH for at least 3 h. The effect of a saturating concentration of GH is qualitatively and quantitatively similar to that produced by 2-4 ng/ml insulin but differs from that of insulin in the respect that adipocytes become refractory to prolonged or repeated stimulation with GH. Since activation of tyrosine kinase is an early event in the action of both hormones, we investigated the possibility that GH stimulation of tyrosine phosphorylation of some protein in the insulin transduction cascade might result in the similar effect of the two hormones. Adipocytes were preincubated for 3 h in the absence of hormones and then reincubated without or with 500 ng/ml GH or 4-400 ng/ml insulin for 10 min. The cells were lysed with an equal volume of buffer containing 1% SDS and preheated to 100 degrees C. Proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes, and tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antiserum coupled to horseradish peroxidase and reagents to produce chemiluminescence. The faint band seen at 185 kDa in control lanes was increased by GH treatment in five independent experiments. Insulin produced a similar effect at a concentration of 4 ng/ml, and phosphorylation increased in a dose-related manner in cells treated with higher concentrations of insulin. A prominent approximately 95-kDa band that is probably not the beta subunit of the insulin receptor was also seen in GH-treated cells. The beta subunit of the insulin receptor has similar electrophoretic mobility to the 95-kDa protein, but was not phosphorylated to an extent that allowed detection when insulin was added at concentrations below 400 ng/ml. Phosphorylation of the 185- and 95-kDa bands was evident within 1 min after addition of GH, persisted for at least 30 min, and was equally prominent in sensitive and refractory cells. Antiserum to IRS-1 immunoprecipitated the tyrosine-phosphorylated 185-kDa protein. The data suggest that IRS-1 is a substrate for a GH-activated tyrosine kinase, possibly JAK-2, which may account for the insulin-like effects of GH. The data further suggest that refractoriness to insulin-like stimulation by GH may result from an additional GH-dependent action that is distinct from phosphorylation of IRS-1.
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PMID:Growth hormone stimulates tyrosine phosphorylation of insulin receptor substrate-1. 752 25

Amphiregulin (AR) is a growth regulatory glycoprotein with significant amino acid homology to members of the epidermal growth factor (EGF) family. Its effects are mediated via the EGF receptor tyrosine kinase or through specific nuclear targeting sequences. In this study, the localization of immunoreactive AR was examined in paraformaldehyde-glutaraldehyde fixed, paraffin-embedded human placentae and decidua obtained at various stages from 11 weeks to term pregnancy using the avidin-biotin complex/peroxidase method. In addition, the effects of AR on trophoblast proliferation were evaluated from 3HTdR uptake by first trimester human trophoblast cells. Results revealed immunolocalization of AR to the nucleus as well as the cytoplasm of the syncytiotrophoblast cell layer of chorionic villi until approximately week 18 of gestation after which no immunostaining was detected. Villous and extravillous cytotrophoblast cells as well as decidual tissue were negative for AR at all gestational ages examined. In the presence of exogenous AR, there was a dose-dependent increase in proliferation of the trophoblast at AR concentrations ranging from 1-100 ng/ml. These results suggest that AR may be an important paracrine or juxtacrine growth stimulatory molecule for cytotrophoblast cells in situ early in gestation.
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PMID:Localization of amphiregulin in the human placenta and decidua throughout gestation: role in trophoblast growth. 756 98

Biochemical studies have shown that basic fibroblast growth factor (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to 15 min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.
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PMID:Basic fibroblast growth factor (FGF-2) internalization through the heparan sulfate proteoglycans-mediated pathway: an ultrastructural approach. 775 May 19

The objective of this work was to investigate the role of tyrosine kinase in monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystal-induced neutrophil activation using the tyrosine kinase inhibitor lavendustin C (LVC). Human neutrophils pretreated with LVC at concentrations between 10 and 150 microM or control neutrophils were stimulated by plasma-coated CPPD or uncoated MSUM, and chemiluminescence, superoxide generation, intracellular calcium concentration, and degranulation (myeloperoxidase and lysozyme release) were monitored with time. LVC strongly inhibited chemiluminescence, superoxide anion generation, myeloperoxidase and lysozyme release, and calcium mobilization. After 1-min crystal-neutrophil incubations, neutrophil cytosolic fractions showed extensive inhibition of tyrosine kinase activity by LVC. We conclude that the inhibition of neutrophil responses to crystal stimulation, by the protein tyrosine kinase inhibitor LVC, provides evidence that supports the involvement of tyrosine kinases in crystal-induced neutrophil activation.
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PMID:Inhibition of crystal-induced neutrophil activation by a protein tyrosine kinase inhibitor. 828 35


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