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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Autoantibodies to thyroid antigens in serums from 34 clinically hypothyroid dogs were detected by various methods. Antibodies to thyroglobulin were detectable by enzyme-linked immunosorbent assay (ELISA) in 59%, by chromic chloride hemagglutination in 53% and by indirect immunofluorescence on formalin-fixed, paraffin-embedded, trypsin-digested thyroid tissue in 73% of samples. Antibody to thyroid microsomal antigen was detectable by ELISA in 29% of serums. Indirect immunofluorescence showed cytoplasmic fluorescence of thyroid follicular cells in several serums, however, this could not be confirmed as specific for microsomal antigen by absorption trials. Hemagglutination tests using commercially available tanned red cells coated with human antigens and indirect immunofluorescence assays on formalin-fixed tissue without trypsin digestion, on Bouin's fixed tissue, or on cryostat, methanol-fixed sections, were insensitive. Cryostat sections without methanol fixation were unsuitable due to tissue fragility. No method was recommended for routine diagnostic use. The ELISA test, because of its convenience, may be useful as a screening aid or adjunct to the diagnosis of thyroid disease.
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PMID:The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, hemagglutination and indirect immunofluorescence. 638 78

Macrophage uptake and presentation of thyroglobulin (TG) were investigated in rats with experimental autoimmune thyroiditis induced by immunization with TG and adjuvant. Peritoneal macrophages were shown to take up radiolabelled TG and this did not depend on the presence of thyroiditis in the animal or on the strain of rat used. Cell-associated antigen was largely unaffected by trypsin. Macrophages primed in vitro with TG produced a rise in the circulating TG autoantibody levels of convalescent animals when given intravenously (unlike free antigen), but did not induce disease in virgin animals. These findings show that macrophages can bind and present TG and suggest that this process may perpetuate otherwise transient autoimmune reactions.
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PMID:Thyroglobulin uptake and presentation by macrophages in experimental autoimmune thyroiditis. 661 16

The orientation of thyroid peroxidase in hog thyroid microsomes was studied by trypsin treatment, gel filtration, binding to Concanavalin A Sepharose and iodination of thyroglobulin. Trypsin treatment of microsomes did not solubilize the thyroid peroxidase activity completely but solubilized the NADPH-cytochrome c reductase activity almost completely. The apparent molecular size of thyroid peroxidase was not altered by trypsin treatment of microsomes. It was, however, decreased by the same treatment of deoxycholate-treated microsomes. On the other hand, the apparent molecular size of NADPH-cytochrome c reductase was reduced by trypsin without prior deoxycholate treatment. Thyroid peroxidase of microsomes did not bind to Concanavalin A Sepharose. Thyroglobulin added exogenously was not iodinated by microsomes, but endogenous thyroglobulin, which had been associated with microsomes, was iodinated. Similar results were obtained with rough microsomal membranes prepared from crude microsomes by sucrose density gradient centrifugation. These results suggest that thyroid peroxidase is oriented toward the luminal side of the microsomal vesicles.
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PMID:Orientation of thyroid peroxidase in hog thyroid microsomes. 661 7

From the cyanogen bromide (CNBr) treatment of porcine thyroglobulin a peptide of mol. wt. 15 000, CNBr-b1, was purified by gel filtration and ion-exchange chromatography. CNBr-b1 contained 50% of the thyroxine (T4) content of the protein. After digestion with trypsin and protease from Staphylococcus aureus V-8, thyroxine-containing peptides were purified and analyzed by microsequence analysis using the colored Edman's reagent dimethylaminoazobenzeneisothiocyanate . Two different sequences harboring T4 were identified: sequence 1, His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-Tyr-Phe-Ser-Ser-Arg, which contains 1 mol T4/mol peptide and sequence 2, Asp-(Tyr/MIT/DIT/T4)-Phe-Ile-Leu-X-Pro-Val-, which is a mixture of the same peptide at different levels of iodination and coupling. These sequences are likely to be representative of distinct hormonogenic sites, the former giving evidence of early iodinated tyrosine residues where preferential coupling into hormonal residues occurs especially at low iodine levels and the latter representing less reactive site(s) operative at higher iodine levels.
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PMID:Structure-function relationship in thyroglobulin: amino acid sequence of two different thyroxine-containing peptides from porcine thyroglobulin. 718 47

The mixture of CNBr peptides obtained by treatment of porcine thyroglobulin with cyanogen bromide was separated into three fractions by Sephadex G-200 gel filtration in 1 M propionic acid. When one of these fractions was reduced and S-alkylated, a hormone-containing CNBr peptide was purified by filtration on Biogel A-1.5 m and ion-exchange chromatography on DEAE-Sephadex. Similarly, two other hormone-containing CNBr peptides were separated from another reduced and S-alkylated fraction by Sephacryl S-200 gel filtration and DEAE-Sephadex chromatography. The three purified CNBr peptides have the same Mr (15000), differ in amino acid composition and contain 60-70% of the hormones present in the initial thyroglobulin. Fragmentation of these peptides into smaller hormone peptides was carried out by trypsin digestion followed by gel filtration. This resulted in the purification of eight discrete hormone-containing tryptic peptides whose homogeneity was established by the study of their N-terminal and C-terminal sequences. At least four sites for the synthesis of thyroxine and two sites for the synthesis of triiodothyronine have been identified in the three CNBr peptides. A possible additional site was recovered in the form of the peptides seryl-triiodothyronine and probably seryl-thyroxine. To gain information on the number of hormone-forming sites in thyroglobulin iodinated in vivo and on the distribution of these sites in the different hormone-containing peptides isolated, estimation of thyroxine and triiodothyronine content of batches of thyroglobulin of iodine content comprised between 0.73% and 1.6% was carried out, together with the estimation of the hormone content of all the CNBr peptides separated from two preparations of thyroglobulin of different iodine contents (0.73% and 1.23%). It was shown that the number of thyroxine and triiodothyronine residues increased linearly with increasing thyroglobulin iodine content in the range 0.73-1.6% and reached six residues of thyroxine and two residues of triiodothyronine/mol protein for a thyroglobulin iodine content of 1.6% with no indication of saturation. This result is at variance with previous findings on porcine thyroglobulin iodinated in vivo which suggested that the thyroxine content reached a plateau of three to four residues/molecule for an iodine content comprised between 0.7% and 1.1%.
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PMID:An approach to the structure of thyroglobulin. Hormone-forming sequences in porcine thyroglobulin. 743 90

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37 degrees C using non-reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides, the largest of which with an apparent molecular weight (MWap) of 40 kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18-24 h of incubation. Tryptic peptides of Tg, released at 1 h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti-Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWap of 10-25 kD and above. Four other MoAbs reacted with peptides of MWap of 25-43 kD and above, and the remaining four reacted with peptides of MWap > 43 kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation-dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.
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PMID:Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies. 752 42

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4 h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15 kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137C1 by these sera. In addition, MoAb 137C1 and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137C1-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.
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PMID:Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases. 752 43

Fifty serum samples from dogs with clinical signs of hypothyroidism and autoantibodies (AA) to thyroglobulin (Tg), thyroxine, or triiodothyronine were screened for AA to thyroid peroxidase (TPO). Thyroid peroxidase is the antigen against which microsomal AA are formed in human beings with lymphocytic thyroiditis. The TPO was isolated from canine thyroid tissue, using a modification of the procedure for purifying porcine TPO. The enzyme was solubilized from the membrane, using a deoxycholate-trypsin solution, followed by ammonium sulfate precipitation and diethylaminoethyl Sephadex chromatography. Activity of TPO was determined, using an iodide oxidation assay and a guaiacol assay. A monoclonal antibody to canine Tg, coupled to an immunoaffinity column, was used to eliminate the contaminating Tg from the TPO preparation. Using the TPO preparation as an antigen, an ELISA was performed on 10 serum samples and immunoblot assays were performed on 50 canine sera. Autoantibodies to TPO were not found in any of the sera. Assays also were performed, using purified porcine and human TPO and evidence of cross-reactivity with canine TPO was not identified. The absence of AA to TPO in dogs suggests a different pathogenesis for autoimmune thyroid disease in dogs than that hypothesized for lymphocytic thyroiditis in human beings.
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PMID:Isolation of thyroid peroxidase and lack of autoantibodies to the enzyme in dogs with autoimmune thyroid disease. 769 46

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.
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PMID:Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells. 802 1

Upon incubation of bovine thyroglobulin with trypsin at high enzyme/substrate ratio, some fragments with apparent masses between 18 and 31 kDa resisted prolonged digestion. Their NH2-terminal sequences were determined. All fragments overlapped with some of the Cys-rich repeats that compose a large part of thyroglobulin and are predicted to have a rigid structure. Among the inserts that interrupt the cysteine-rich repeats, for which several data indicate a location at the surface of thyroglobulin, the insert of repeat 1.7 was unique because of its resistance to proteolysis. This insert, although exposed at the surface of the protein, may be hidden in a region of contact between thyroglobulin monomers.
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PMID:Trypsin-resistant regions of thyroglobulin: possible relationship with intermonomeric contact site(s). 825 Aug 70


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