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The mRNA encoding human thyroglobulin has been cloned and sequenced. It is made up of a 8301-nucleotide segment encoding a preprotein monomer of 2767 amino acids, flanked by non-coding 5' and 3' regions of 41 and 106 nucleotides, respectively. This preprotein consists of a leader sequence of 19 amino acids, followed by the sequence of the mature monomer, corresponding to a polypeptide of 2748 amono acids (Mr = 302773). On its amino-terminal side, 70% of the monomer is characterized by the presence of three types of repetitive units. In contrast, the remaining 30% of the protein is devoid of repetitive units. This last region however shows an interesting homology (up to 64%) with the acetylcholinesterase of Torpedo californica. The sites of thyroid hormones synthesis are clustered at both ends of the thyroglobulin monomer. By contrast, the potential glycosylation sites are scattered along the polypeptide chain. [ABSTRACT FROM AUTHOR]

Thyroglobulin, the dimeric glycoprotein (19 S, 2 × 330 kDa), specific to the thyroid gland, is the support for thyroid hormone synthesis. Elucidution of the mechanism for thyroid hormone synthesis requires the knowledge of the primary sequence of the protein. In this paper the sequence of the first coding 2190 nucleotides from the 5′ end of the human mRNA is presented. This was obtained by sequencing two previously described overlapping clones and by construction and sequencing of a single-stranded cDNA corresponding to the 5′ end of the mRNA. The nucleotide sequence represents a quarter of the human thyroglobulin mRNA, from which a polypeptide sequence of 730 amino acids at the NH2-terminal end of the monomer has been deduced. This sequence shows u repetition of five highly conserved motifs each of approximately 50 amino acids, the analysis of which allowed us to establish a consensus sequence. We have also demonstrated (a) the hormonogenic tyrosine residue recently described in the mature protein, which is located four amino acids alter the NH2-terminal ASn: (b) a prepeptide signal of thyroglobulin secretion comprising 19 amino acids preceding the Asn residue, the NH2-terminal residue of the mature protein and (c) a six-signal tripeptide (Asn-Xaa-Thr or Ser) of N-glycosylation of the chain. [ABSTRACT FROM AUTHOR]

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The later was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease pstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA · mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63 % of the 8500 bases of human thyroglobulin mRNA. [ABSTRACT FROM AUTHOR]

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 M, (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 dipeptides 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%. [ABSTRACT FROM AUTHOR]

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5–50 μU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 μM) augmented thyrotropin stimulation and dibutyryl adenosine 3′:5′-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators. [ABSTRACT FROM AUTHOR]

Isolated porcine thyroid cells, cultured in the presence of thyrotropin (≥ 0.25 mU/ml) or prostaglandin E2 (≥ 0.1 μM), showed decreased adenosine 3′: 5′-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin E2 stimulation, respectively. Kinetics of the refractory process to thyrotropin and prostaglandin E2 are different: (a) maximal refractoriness to prostaglandin E2 was attained after 2–6 h exposure to prostaglandin E2 while refractoriness to thyrotropin was maximal only after 12–24 h; (b) the degree of refractoriness to prostaglandin E2 was much greater than that to thyrotropin. Refractoriness to thyrotropin or prostaglandin E2 is characterized: by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 μM prostaglandin E2), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility. This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3′:5′ monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin E2 by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state. [ABSTRACT FROM AUTHOR]

The time course of digestion of porcine thyroglobulin with subtilopeptidase at an enzyme to substrate ratio of 1 :200 was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis after reduction with dithiothreitol. Two fragments of Mr 155000 and 135000 were formed which showed a precursor-product relationship, the 135000-Mr species accumulating between 1 and 4h of digestion. After purification by elution from electrophoretic gels, these fragments were treated by CNBr and analyzed by dodecylsulfate/poiyacrylamide gel electrophoresis. Both mixtures of CNBr peptides showed a closely similar electrophoretic band pattern. These results suggest the presence in the thyroglobulin chain (Mr 330000) of half-sized segments each formed of a domain with strong tertiary structure of Mr about 140000 associated through the continuity of the chain to a less structured fragment of Mr about 20000. A 15000-Mr peptide was purified by Sephadex G-200 gel filtration from CNBr-treated thyroglobulin. Its uniqueness was demonstrated by N-terminal sequence analysis and by tryptic fingerprinting. Using densitometer recording of stained electrophoretic gels in sodium dodecylsulfate, the molar ratio of this peptide was estimated to be 2 mol/mol of 12-S thyroglobulin chain. These results are consistent with a protein structure in which two domains of similar size and structure are linked to form a single chain. Thus, the possibility exists that the structural gene for thyroglobulin might have evolved from duplication and fusion of an half-sized ancestral gene. [ABSTRACT FROM AUTHOR]

Undegraded 19-S thyroglobulin was purified from hog, ox, man, dog, sheep and rat thyroid glands. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the reduced proteins showed that all are formed of peptide chains of molecular weight about 330 000. Carboxypeptidase A digestion of porcine 19-S thyroglobulin released consecutively two moles of leucine and then two moles of serine, thus offering strong evidence in favour of the idea that the protein is formed of two identical chains. The same C-terminal amino acids were detected in sheep, ox, dog and man thyroglobulins. No N-terminal amino acid was found by appropriate chemical and enzymatic techniques. Porcine 27-S iodoprotein was shown by carboxypeptidase A analysis to be formed of four single-stranded 330 000-Mr subunits identical to those constituting the 19-S protein. Identity, both qualitatively and quantitatively, of the peptides obtained by CNBr cleavage of the two proteins as shown by sodium dodecylsulfate/polyacrylamide gel electrophoresis has confirmed this conclusion. Since 19-S and 27-S thyroid iodoproteins are formed of two and four probably identical chains, they must be termed 19-S and 27-S thyroglobulins or alternatively thyroglobulin dimer and tetramer, respectively. [ABSTRACT FROM AUTHOR]

Total polysomes were obtained by homogenization of sheep thyroid glands in Tris-Cl-buffered sucrose, pH 7.5, treatment with 2% Triton X-100 and centrifugation of the magnesium-treated 27000 × g supernatant through 1 M sucrose. Purification of thyroglobulin-specific polysomes was performed by indirect immunoprecipitation with rabbit antibodies to sheep thyroglobulin and donkey antibodies to rabbit immunoglobulin G. Thyroglobulin polysomes represented 30% of total polysomes. Synthesis of thyroglobulin peptides in a reticulocyte cell-free system, programmed with total RNA from immunoprecipitated polysomes, was 3 times higher than when total RNA from unfractionated polysomes was used. The size of the poly(A)-containing thyroglobulin mRNA was estimated by the capacity of RNA fractions obtained by sucrose density gradient centrifugation of total or immunopurified polysomal RNA to hybridize with ³H-labeled poly(U) and to stimulate thyroglobulin peptide synthesis in a reticulocyte lysate. 65% of the thyroglobulin messenger activity was located in a 33–36-S RNA peak and 35% in zones corresponding to 30-S and 26-S RNA species, The thyroglobalin-specific immunoprecipitable peptides synthesized in the reticulocyte lysate from the 33–36-S RNA were shown by polyacrylamide gel electrophoresis in denaturing conditions to contain a major component co-migrating with the constituent peptide chain of sheep thyroglobulin (Mr 330000). Other components migrated with Mr of about 210000 and 120000. [ABSTRACT FROM AUTHOR]

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the β subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 °C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine > porcine > human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones. [ABSTRACT FROM AUTHOR]

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physicochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 μg). [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [ABSTRACT FROM AUTHOR]

Plasma membranes have been prepared from porcine thyroid cells stimulated by dibutyryl adenosine 3′:5′-monophosphate in culture. These cells show the histiotypic organization in follicles and the metabolic properties of gland follicular cells. The plasma-membrane fractions have a density in sucrose of 1.18 g/ml (about 80%) and 1.16 g/ml (about 20%). The yield is 2 mg per 6 × 108 cells. Electron microscopy showed vesicular structures and sheets of membranes often joined by membrane junctions. Both showed the triple layer characteristic of the unit membrane ultrastructure. Less than 3% of contamination by microsomes was derived from the assay of NADPH-cytochrome c reductase and NADH oxidase. No contamination by mitochondria was indicated from succinate dehydrogenase activity measurements. NaF- and thyrotropin-stimulated adenylate cyclase, 5′-nucleotidase and alkaline phosphatase are concentrated in the plasma-membrane preparations. Specific binding of 125I-labeled thyrotropin of high specific radioactivity (2.5 Ci/μmol) and preserved biological activity was compared to that in intact cells and to the plasma membranes from which they derived. The rate constants of the thyrotropin association with cell or membrane (0.56 and 0.11 nM-1 min-1, respectively) and dissociation (0.36 and 0.25 min-1, respectively) were measured independently at 35 °C (cells) and 27 °C (membranes). The association constants derived from these rate constants (1.5 and 0.4 nM-1 for cells and membranes) are very similar to that (1.9 and 0.6 nM-1) determined independently from equilibrium data. Therefore plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation. The relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotropin. The semilogarithmic plot of thyrotropin concentration correlates sigmoidally with the percentage of adenylate cyclase activation. Half-maximum stimulation is obtained for a thyrotropin concentration of 1.8 nM which correlates remarkably with the dissociation constant of 1.78 nM determined for the binding of the hormone to membranes. [ABSTRACT FROM AUTHOR]

The thyrotrophin-induced reorganization of isolated porcine-thyroid cells into follicles is specifically mediated by cyclic AMP. Both dibutyryl cyclic AMP and theophylline at concentrations of 0.1 mM and 0.05 mM, respectively, mimic the effect of thyrotrophin. Puromycin and cycloheximide acutely inhibit the cyclic AMP-induced histiotypic reassociation of cells. Actinomycin-D inhibits cell organization when added at zero-time or 12 h after the onset of the culture but not when added 24 h later. Therefore, the intracellular increase in cyclic AMP is most likely a prerequisite to the reassociation into follicles of isolated thyroid cells. Cyclic AMP stimulates the production of new RNA(s) which, in turn, induces the synthesis of protein(s) involved in intercellular recognition. Isolated thyroid cells grow as a monolayer in the absence of thyrotrophin or cyclic AMP. Between the first and the second day of culturing, they lose the capacity to concentrate iodide. From the third to the thirteenth day neither iodide trapping nor organification can be detected. In contrast, cells organized into follicles maintain their capacity to concentrate iodide, iodinate thyroglobulin and synthesize thyroid hormones. Quantitative estimation of thyroglobulin was performed by radioimmunoassay. Up to the seventh day of culture, the total amount of thyroglobulin recovered per cell in the medium and in the cell layer is roughly identical whether the cells are cultured as a monolayer or in histiotypic reassociation. Non-iodinated thyroglobulin is secreted into the medium by monolayer cells whereas 19-S iodinated-thyroglobulin is found in the medium of thyrotrophin-induced reassociated cells. We therefore conclude that thyrotrophin is necessary to maintain the differentiation of isolated thyroid cells in culture as judged by the preservation of such specific functions as the capacity to aggregate and reorganize into follicles, to concentrate and organify iodine and to synthesize thyroid hormones. [ABSTRACT FROM AUTHOR]

Porcine thyroid cells isolated by trypsinization grow in culture as a monolayer. At high cell concentration and in the presence of thyrotrophin, cells aggregate and rearrange in structures resembling cross-sections of intact thyroid tissue. Longitudinal and transverse sections of thyrotrophin-stimulated and non-stimulated cell cultures have been examined by electron microscopy after fixation of the cell layers in the culture flasks. Cells cultured in the presence of thyrotrophin undergo an organization rearrangement into typical three-dimensional follicles as shown by the presence of junctional complexes, differentiated apical poles with microvillae, follicular lumen sometimes containing material dense to electrons, and apical vesicles. In contrast, cells cultured in the absence of thyrotrophin develop as a two-dimensional layer. No differentiation of the plasma membrane is visible. Cells are flat and partially overlap each other. All cells are linked by junctional complexes. Except for this zone of association, the undifferentiated plasma membranes of the contiguous cells are not adherent and limit more or less distended intercellular spaces. Cells associated in follicles show the ultrastructural aspects of polarized and stimulated follicular cells. The apical pole is rich in organelles such as lysosomes, vesicles, Golgi complexes and rough endoplasmic reticulum. To the contrary in monolayer cells, the organelles are perinuclear; Golgi complexes are scarce and a very dense network of microfilaments and microtubules is visible in the peripheral part of the cytoplasm. Therefore, thyroid cells cultured with or without thyrotrophin, each form a homogeneous population of cells in a given organization arrangement and at different level of differentiation. [ABSTRACT FROM AUTHOR]

Using the same method of purification of small molecular weight proteins worked out to obtain pure scorpion neurotoxins, four neurotoxins have been isolated from the venom of the cobra Naja haje. N. haje venom from the Miami Serpentarium contains two neurotoxins (I and II) whereas the venom of the same species obtained from the Institute Pasteur contains three neurotoxins (I, II' and III). Toxins I, II and II' consist of 61 amino acid residues and toxin III of 71. All are basic proteins containing four or five disulphide bridges. It is likely that the difference observed in neurotoxin composition of N. haje venoms from two different sources has a taxonomical counterpart, each venom originating from a different sub-species. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of neurotoxin I of the North African scorpion Androctonus australis Hector collected in Algeria (area of Chellala) is described. S-Methylation of cysteine residues formed by reduction of disulphide bonds was found useful for their identification in sequence determination using the phenylisothiocyanate method. From the N-terminal sequence (38 residues) established in the protein sequenator, and from the sequence of chymotryptic peptides the complete sequence of the protein has been deduced. The molecule consists of a single polypeptide chain cross-linked by four disulphide bridges. An isoneurotoxin (I') has been found in the venom of A. australis collected in Tunisia (area of Tozeur). It differs from neurotoxin I in the replacement, of the valine residue in position 17 by an isoleucine residue. [ABSTRACT FROM AUTHOR]