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Title: Studies on thyroidal protein biosynthesis in relation to thyroid hormone biosynthesis
Author: Fairley, C. J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1969
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1. The object of the study was the isolation from thyreoglobulin of peptides containing iodothyronines or iodotyrosines, followed by investigation of these peptides as the actual or potential sites of thyroid hormone synthesis in thyreoglobulin. 2. A cell free preparation capable of incorporating amino acids into protein was isolated from rat thyroid. Some of the unusual properties of this preparation were studied. 3. Thyroglobulin containing ¹⁴C-amino acids was isolated from whole rat thyroids after Incubation with ¹⁴C-amino acids. Under similar conditions sheep thyroid slices rapidly incorporated ¹⁴C- amino acids and ¹³¹I⁻ yielding thyroglobulia highly labelled with both isotopes. 4. Thyroglobulin isolated from sheep thyroid slices was purified, by fractionation with ammonium sulphate or by gel filtration on Sephadex G-200. The purity of the preparation was determined by a variety of methods including gel filtration, electrophoresis and ultraoentrifugation. Suorose gradient centrifugation revealed a highly iodinated lighter protein fraction. 5. Labelled thyroglobulin was hydrolysed by α-chymotrypsin and the resulting hydrolysate subjected to electrophoresis followed by chromatography at right angles (peptide mapping). Autoradiograms of these peptide maps revealed the presence of a number of peptides labelled with ¹³¹I₂ and a larger number labelled with ¹⁴C-tyrosine. None of the ¹³¹I-peptides was unequivocally labelled with ¹⁴C- tyrosine. 6. The most highly labelled ¹³¹ I-peptides were isolated, in some cases purified by electrophoresis at pH 8.2, and their iodoamino acid contents found after pronase hydrolysis and chromatography. Each peptide contained only one iodotyrosine confirming the specific nature of tyrosyl iodination indicated by the small number of intensely labelled ¹³¹I-peptides compared with a larger number of ¹⁴C-tyrosine peptides. 7. The approximate sizes of the ¹³¹I-peptides were determined by estimation ©i the bonds hydrolysed by αC-chymotrypsin and by gel filtration of the peptides on Sephadex 0-25. 8. During the incorporation of ¹³¹I⁻ into thyroid slices over a period of 5 hr. the activity of each ¹³¹I-peptide, as a percentage of the total ¹³¹I₂ in thyroglobulin, did not alter although the ratio of the ¹³¹I-activities of mono- to diiodotyrosine fell throughout this period. 9. Monoiodotyrosyl peptide A₅ was iodinated chemically with ¹³¹I₂. The product was not identical with already isolated diiodotyrosyl peptides of similar mobilities on peptide mapping. 10. Peptide A₅ was associated with another peptide (separated by electrophoresis at pH 8.2) whose products of iodination were identical with those of A₅ and which, it is suggested, is the unlodinated form of A₅. 11. This latter peptide taken together with A₅, is present as 4 moles per mole of thyroglobulin as determined by iodination with 131I₂ of known specifio aotivity. Two other peptides, N₉ and N₉ₐ' are each present as 5 moles per mole of thyroglobulin. It ya is suggested that this confirms the evidence that thyroglobulin oomprises four identioal sub-units and that iodotyrosyl residues can remain partially uniodinated as well as being mono- or diiodinated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available