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
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-
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.
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.