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:1.11.1.8 (thyroid peroxidase)
3,116 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In contrast to other studies, our results demonstrate that low concentration of trypsin degrades a high proportion of proteolipid from CNS myelin. The Wolfgram protein and BP are vulnerable and completely lost on trypsinolysis, perhaps accounting for some of the peptides retained by the myelin. In PNS myelin, the major PO protein, a hydrophobic glycoprotein, is readily degraded to a stable 18,000--19,000 molecular weight unit, referred to as TPO protein, still retaining the carbohydrate unit which probably exists as a nonasaccharide grouping. Production of the TPO glycoprotein results from cleavage of a lysinyl-methionine or arginyl-methionine linkage probably found approximately 80--100 residues from the NH2-terminal isoleucine of the PO molecule. This linkage must be especially accessible to trypsin since the TPO protein is also generated in high yield when isolated PO protein is treated with trypsin in solution for 0.5 hours. Further incubation for 24 hours fully degrades the TPO protein to over 20 tryptic peptides, shown by peptide mapping, unlike the situation in myelin where the TPO unit is stable and resists further proteolysis. The TPO unit is also produced when PO protein is treated with BrCN. The PO protein contains 3 methionine residues but presumably the methionine residue in the trypsin-sensitive region is crucial; cleavage leads to the same TPO unit minus NH2-terminal methionine. Another methionine residue also exists in the TPO protein but it may be resistant to BrCN cleavage or else occupy a near-end position. Other proteins were also identified on PAGE of trypsinized PNS myelin: albumin, P2 protein, and PO protein. Albumin and P2 protein were identified in the acidic extract by reaction with specific antibody. The PO protein was isolated; it moved similarly to standard protein on SDS-PAGE and gave the appropriate amino acid analysis. However, it cannot be determined at this time whether a portion of these proteins remains because they are partially inaccessible to trypsin, or else are slightly attacked and thus represent early stages of trypsinolysis. The P2 protein of trypsinized myelin appears to migrate slightly faster than standard P2 protein on PAGE. Further work should clarify this point. Amino acid analysis and sequence data show that the PO protein is particularly hydrophobic, very likely existing in PNS myelin as an amphipathic molecule which penetrates the bilayer but which has a hydrophilic portion exposed. It is this hydrophilic region that contains much lysine, particularly the crucial lysinyl-methionine linkage, that is so trypsin-sensitive. Determination of the amino acid sequence of terminal portions of the isolated PO and TPO proteins serves to firmly establish the PO protein as a unique entity probably exclusive to PNS myelin. It can be concluded that the study of trypsin activity toward PNS myelin has made possible a new understanding of how proteins are positioned in the membrane, and provided valuable insight into the PO protein.
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PMID:The action of trypsin on central and peripheral nerve myelin. 69 76

Two patients (G2, G3) with iodine organification defect were studied. The first patient (G2), a 25-year-old women with no clinical hypothyroidism, had had her goiter for 10 years; 62% of the thyroidal iodine was released by perchlorate indicating iodine organification defect. The thyroid tissue obtained at thyroidectomy contained a normal concentration of thyroid peroxidase (I2 formation from I-) when tested after solubilization of the enzyme by trypsin and digitonin treatment of the particulate material. 1. The enzymatic activity (G2-TPO) behaved on DEAE cellulose chromatography very differently from those of hog (P-TPO) or another human goiter peroxidase (G1-TPO) (Pommier, et al., J Clin Endocrinol Metab 39: 69, 1974): the molarity of elution was 2M NaCl instead of 0.15 mM. 2. Both P-TPO and G2-TPO catalyzed iodide peroxidation (I- leads to I2) but the Km (iodide) value for G2-TPO was much lower (2.3 x 10(-2) M) when compared with that of P-TPO (3.7 x 10(-3) M) or G1-TPO (3.5 x 10(-3) M). In addition, the optimum pH for this reaction differed markedly (pH 6.1 instead of 7.9). 3. G2-TPO was poorly efficient in catalyzing the oxidation of gaiacol to tetragaiacol. 4. G2-TPO was unable to perform the iodination of non-iodinated goiter thyroglobulin whatever the pH and the iodide concentration. 5. Thyroglobulin from this goiter (G2) was almost not iodinated (0.0014%), i.e., 0.07 atoms iodine/mole thyroglobulin), and its total content in the gland was very low (0.3-4 g/1000 g wet tissue instead of 25 g). A clear discrepancy was thus shown between the euthyroid state of this patient and the total lack of iodinating activity of the isolated peroxidase. The second patient (G3), a 17-year-old man with clinical hypothyroidism, had had his goiter for 5 years. 100% of the thyroidal iodine was released by perchlorate indicating a complete iodine organification defect. The thyroid tissue obtained at thyroidectomy contained no peroxidase activity when tested before and after treatment of the particulate material by trypsin and digitonin and even in the presence of hematin. Thyroglobulin from this goiter, which was almost non-iodinated (0.0014%), was present in normal amounts in the gland (congruent to 25 g/1000 g).
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PMID:Thyroid iodine organification defects: a case with lack of thyroglobulin iodination and a case without any peroxidase activity. 126 32

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.
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PMID:In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells. 131 Jun 79

Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC). The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E. coli. A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins. The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA). Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R1a + b (residues 1-160) and more specifically, in the case of MoAb A4, within the subfragment R1b (residues 70-160). The inability of the other MoAbs (A1-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins.
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PMID:Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations. 137 60

The cortex of the upper bank of the superior temporal sulcus (STS) in the rhesus monkey contains a region that receives overlapping input from post-Rolandic sensory association areas and is considered multimodal in nature. We have used the fluorescence retrograde tracing technique in order to answer the question of whether multimodal areas of the STS project back to post-Rolandic sensory association areas. Additionally, we have attempted to answer the question of whether the projections from the multimodal areas directed to the parasensory association areas originate from common neurons via axon collaterals or from individual neurons. The results show that multimodal area TPO of the STS projects back to specific unimodal parasensory association areas of the parietal lobe (somatosensory), superior temporal gyrus (auditory), and posterior parahippocampal gyrus (visual). In addition, a substantial number of projections from area TPO are directed to distal parasensory association areas, area PG-Opt in the inferior parietal lobule, areas Ts1 and Ts2 in the rostral superior temporal gyrus, and areas TF and TL in the parahippocampal gyrus. These latter regions are themselves considered to be higher-order association areas. It was also noted that the majority of the projections to these higher-order association areas originate from the middle divisions of area TPO (TPO-2 and TPO-3). These neurons are organized in a significantly overlapping manner. Despite this overlap of the projection neurons, only an occasional double labeled neuron was observed in area TPO. Thus, our observations indicate that the multimodal region of the superior temporal sulcus has reciprocal connections with the unimodal parasensory association cortices subserving somatosensory, auditory and visual modalities, as well as with other post-Rolandic higher-order association areas. These connections from area TPO to post-Rolandic association areas may have a modulating influence on the sensory association input leading to multimodal areas in the superior temporal sulcus.
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PMID:Efferent cortical connections of multimodal cortex of the superior temporal sulcus in the rhesus monkey. 158 61

Carboxyl terminal truncation of membrane-associated human thyroid peroxidase (hTPO), with the elimination of its single membrane-spanning and short intracytoplasmic regions, generates a soluble, secreted, enzymatically active protein (amino acids 1-848). In order to determine the effects of further carboxyl terminal deletions on the expression of hTPO, Chinese hamster ovary cells were stably transfected with plasmids constructed to express amino acids 1-771, 1-636, 1-539 and 1-382 of the 933 amino acid TPO protein, respectively. Unlike hTPO1-848, the more severely truncated TPO mutant proteins could not be detected in conditioned media by polyclonal anti-TPO antibodies. Using detergent-solubilized microsomal proteins from these cells, very low levels of hTPO1-771 (approximately 90 kDa), but not the more extensive deletion mutations, were detected by these anti-TPO antibodies. Confirmation of the loss of efficient expression of more severely truncated hTPO was obtained using a anti-hTPO monoclonal antibody with an epitope near the amino terminus and which recognizes only the denatured protein. The mRNA for all hTPO mutants was detected in the stably-transfected Chinese hamster ovary cells. In summary, the present study indicates that a largely intact extracellular portion of hTPO is required for expression in eukaryotic cells.
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PMID:Importance of the carboxyl terminus of human thyroid peroxidase in the efficient expression of the protein in eukaryotic cells. 163 19

Evidence has accumulated in the last few years that the expression of the microsomal/peroxidase antigen (M/TPO-Ag) in thyroid cells is induced by TSH, through pathways which involve intracellular cAMP accumulation and protein synthesis. These data have been found true in any thyroid system studied so far, both in terms of immunologic and enzymatic activity of TPO. TSH and cAMP also increase the levels of the specific mRNA for TPO in thyroid cells from different species. Whether this phenomenon is due to a direct transcriptional regulation of the TPO gene, as shown in dog thyroid cells, or to posttranscriptional effects, as it would appear in FRTL-5 cells, remains to be clarified by future experiments. Thyroid stimulating antibody (TSAb) of Graves' disease also stimulates the expression of M/TPO-Ag. This finding gives further support to the relevance of TSAb in the pathogenesis of hyperthyroidism and explains the well known observation that the "microsomal" antigen is particularly abundant in glands of Graves' patients. The modulation of M/TPO-Ag surface expression by TSH can explain the decrease of circulating anti-MAb observed during L-thyroxine therapy in hypothyroid patients with Hashimoto's thyroiditis. Other agents, such as methimazole and sodium iodide, which influence thyroid cell function, do not directly interfere with the expression of M/TPO-Ag. Cytokines, such as gamma-interferon, interleukin-1, and interleukin-6 have been shown to inhibit the TSH-induced increase of TPO mRNA, but further investigations are required to elucidate the exact role of cytokines in the regulation of M/TPO-Ag expression.
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PMID:The microsomal/peroxidase antigen: modulation of its expression in thyroid cells. 166 95

This paper describes the use of sensitized sheep red blood cells for the detection and titration of complement fixation by autoantibodies directed against human thyroid membranes in the serum of patients with autoimmune thyroid disease. Patients with elevated circulating levels of TPO antibodies and diagnosed as having autoimmune hypothyroidism (including Hashimoto's disease) or autoimmune hyperthyroidism (Graves' disease) were studied. Complement fixation titres were highest in those patients with autoimmune hypothyroidism compared with the autoimmune hyperthyroid group. Serum samples obtained from a group of patients with thyroid neoplasia and from normal healthy volunteers were negative in this test. The TPO antibody activity when "corrected" for its CF potency suggests that the autoantibodies found in autoimmune hypothyroidism are potentially more destructive than those found in the non-destructive autoimmune thyroid diseases.
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PMID:The measurement of complement fixation by autoantibodies directed against thyroid membrane antigens. 166 85

Recombinant, enzymatically active human thyroid peroxidase (hTPO) generated in nonthyroidal eukaryotic cells was compared with Graves' thyroid microsomes as a source of antigen for the immunological detection of antimicrosomal/anti-hTPO antibodies. Enzyme-linked immunosorbent assay of 51 sera, selected to produce a balanced distribution of antimicrosomal antibody (anti-MSA) levels, revealed (at 1:100 serum dilution) a moderately good correlation between anti-MSA and anti-hTPO antibody levels (r = 0.668; P less than 0.001). However, a number of sera with high anti-MSA levels yielded markedly discordant values between the two assays. A much lower correlation was observed between antithyroglobulin and anti-hTPO antibody levels (r = 0.315; P less than 0.05). At higher serum dilutions (1:1,000 and 1:10,000), at which low affinity, high capacity binding reactions will be reduced, the correlation between anti-MSA and anti-hTPO antibody values was greatly improved (r = 0.906 and 0.902, respectively; P less than 0.001), and there were no longer widely discrepant values between the two assays. In summary, the present study indicates that recombinant hTPO expressed in nonthyroidal cells provides an unlimited source of human TPO of unvarying quality for anti-hTPO antibody assays. This material offers increased specificity over standard anti-MSA assays that use thyroid cell microsomes as antigen.
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PMID:Recombinant human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease. 168 38

We studied the oligosaccharide moieties of recombinant human thyroid peroxidase (hTPO) expressed in Chinese hamster ovary (CHO) cells, and the role of these glycans in hTPO antigenicity in Hashimoto's thyroiditis. To determine whether hTPO carbohydrate moieties were N-linked, O-linked, or both, and to obtain information about the characteristics of the carbohydrate component(s), we digested hTPO with deglycosylating enzymes of varying specificity. Proteins in CHO-TPO cells were labeled with [35S]methionine, and hTPO was immunoprecipitated with anti-hTPO antibodies present in Hashimoto's thyroiditis serum. Digestion with endoglycosidase (endo) F, which removes both complex and polymannose N-linked glycans, increased the electrophoretic mobility of the hTPO doublet from approximately 115 kD and 110 kD to 110 kD and 105 kD. Endo H, which acts similarly to endo F, but only on polymannose, and not complex, glycans, had a similar effect. In contrast, O-glycanase and neuraminidase, which remove O-linked glycans and terminal neuraminic acid, respectively, did not alter the mobility of radiolabeled hTPO. Radiolabeled recombinant hTPO was retained by concanavalin A, but not by wheat germ agglutinin, Ricinus communis agglutinin 1, peanut agglutinin and Ulex europaeus lectins. To determine whether or not the glycan moieties in hTPO play a role in the disease-associated epitopes in Hashimoto's thyroiditis, radiolabeled recombinant hTPO was immunoprecipitated after digestion with N-glycanase. Removal of the N-linked carbohydrate chains with endo F and endo H did not prevent antibody binding. In summary, the present data indicate that: i) hTPO expressed in CHO cells contains N-linked, but not O-linked glycan moieties; ii) the N-linked carbohydrate is primarily of the polymannose variety; and, iii) the glycan moieties do not contribute to the hTPO epitopes in Hashimoto's thyroiditis.
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PMID:Carbohydrate moieties in recombinant human thyroid peroxidase: role in recognition by antithyroid peroxidase antibodies in Hashimoto's thyroiditis. 169 64


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