Ann D. Dunn

Tyrosine 130 Is an Important Outer Ring Donor for Thyroxine Formation in Thyroglobulin

The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14C]tyrosine into beef thyroid slices, isolated and iodinated the [14C]thyroglobulin (Tg I), separated its N-terminal ∼22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129. An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130–146 with Tyr130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion. 14C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr5, the most important hormonogenic site of thyroglobulin, because Tyr5 and Tyr130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T4 by itself.

The importance of thyroglobulin structure for thyroid hormone biosynthesis.

Thyroglobulin (Tg) is the most important protein in the thyroid because it provides the matrix for thyroid hormone biosynthesis. Here we review experimental work, principally from our laboratory, on the relationship between Tg structure and hormonogenesis. Early work showed that Tgs most important hormonogenic site was located in a fragment of approximately 26 kDa obtained on chemical reduction. With the establishment of the cDNA sequence of Tg, this and other major sites could be localized within Tgs polypeptide chain. The four major hormonogenic sites, designated A, B, C, and D, are located respectively at tyrosyls 5, 2553, 2746, and 1290. In most species, site A accounts for about 40% of Tgs hormone, and site B for about 25%. Site C is associated with increased T3, at least in some species. Site D is prominent in guinea pigs and rabbits, and TSH favors hormonogenesis at it in these species. Sequential iodination of low iodine human Tg shows three consensus sequences associated with early iodination and with T4 formation. Recent work has identified Tyr130 in beef Tg as donor of an outer iodothyronine ring, most likely to Tyr5, the most important hormonogenic site. In addition to its biochemical importance, Tg has clinical interest in familial goiter and autoimmune thyroid disease. Further elucidation of Tg structure and its relation to thyroid hormone synthesis will contribute to thyroid physiology and to its clinical application.

Fast Colorimetric Method for Measuring Urinary Iodine

International groups recommend the following median urinary iodine concentration as the best single indicator of iodine nutrition in populations: severe deficiency, 0–0.15 μmol/L (0–19 μg/L); moderate deficiency, 0.16–0.38 μmol/L (20–49 μg/L); mild deficiency, 0.40–0.78 μmol/L (50–99 μg/L); optimal iodine nutrition, 0.79–1.56 μmol/L (100–199 μg/L); more than adequate iodine intake, 1.57–2.36 μmol/L (200–299 μg/L); and excessive iodine intake, ≥2.37 μmol/L (≥300 μg/L) (1). The range in which the median falls is more important than the precise number (2)(3). Many methods for assessing urinary iodine exist (3)(4)(5)(6)(7)(8), most based on the Sandell–Kolthoff reaction (9), in which iodide catalyzes the reduction of ceric ammonium sulfate (yellow) to the colorless cerous form in the presence of arsenious acid. Although iodide is the chemical form for both the catalytic reaction and in urine, some preliminary treatment is needed to rid urine of impurities, most commonly by acid digestion (3)(5). We have extended previous approaches (5)(6)(10) with improved conditions and here present a new method (“Fast B”) that is rapid, inexpensive, reliable, and flexible. The equipment required for the Fast B method includes a heating block, Pyrex test tubes (13 × 100 mm), two fixed-volume pipettes (0.5 mL and 1.0 mL), one adjustable pipette (0–200 μL), and a multipet (Eppendorf) for quick reagent volume additions of 0.125 and 0.1 mL. The basic chemicals used are potassium iodate, arsenic trioxide, ammonium persulfate, ammonium cerium(IV) sulfate dihydrate, sodium chloride, ferroine, and sulfuric acid. The solutions used in the assay are as follows:

Properties of an iodinating enzyme in the ascidian endostyle

An iodinating enzyme was extracted from the endostyles of Molgula manhattensis and Styela clava by a combination of surfactant treatment and trypsin digestion. The solubilized enzyme was partially purified by column chromatography. The enzyme behaves as a large molecular complex (MW ∼ 340.000) and is partially hydrophobic in nature. It catalyzes the iodination of tyrosine or protein optimally at a pH of about 7.5. Iodinating activity is inhibited at concentrations of iodide above 0.5 mM. The enzyme is capable of catalyzing the coupling of iodotyrosyl residues in thyroglobulin in vitro, forming T4 and T3. It is suggested that the lack of significant iodothyronine formation in the endostyle in vivo is due to the absence of a suitable protein substrate rather than to an enzymatic deficiency.

Studies on iodoproteins and thyroid hormones in ascidians

Abstract Iodoproteins in the ascidian Molgula manhattensis were labeled in vivo with 125 I. The proteins were extracted with water and surfactants and analyzed by gel filtration and by polyacrylamide gel electrophoresis. They were found to be composed largely of small subunits with molecular weights of approximately 22,000 in the tunic and 14,000–15,000 in the endostyle and pharynx. Radioimmunological evidence was found for small amounts of thyroid hormones in the tunics of Molgula, Ciona intestinalis , and Styela clava . Values of 88–220 ng thyroxine (T 4 )/g of tissue and 8–16 ng 3,5,3′-triiodothyronine (T 3 ) were obtained. Seventy five percent of the T 4 in the tunic was in protein-bound form. Trace amounts of T 4 (3 ng/g) and possibly T 3 were found in the endostyle of Molgula , primarily in free form. Evidence for T 4 was found in the blood of Ciona where it may be noncovalently linked to serum proteins. On the basis of molecular size, the endostylar proteins are distinct from proteins in the surfactant-solubilized fraction of the tunic, and protein subunits at both sites are smaller than those of vertebrate thyroglobulin. Whether the iodothyronines found in the tunic, endostyle, and blood are formed within proteins at each of these sites or are synthesized at one site and then transported to another remains to be determined.

Iodine metabolism in the ascidian, Molgula manhattensis

Abstract Specimens of Molgula manhattensis were immersed in sea water containing 125 I. Light-microscopic autoradiography of the soft tissues revealed that bound iodine was confined to the endostyle and to the lumina of the pharynx and intestines. The presence of iodinated material was investigated in homogenates of the endostyle, extraendostylar pharynx, tunic, and gonads. PB 125 I was found in the tunic, endostyle, and extraendostylar pharynx after exposure to 125 I for 1 or more hr. After long-term exposures (7–10 days), small amounts of PB 125 I were detected in the gonads as well. MIT and DIT, but not T 4 or T 3 , were identified in proteolytic digests of these four tissues. The ratio of DIT:MIT was higher in the endostyle than in the other tissues. Homogenates of the endostyle and tunic, but not of the extraendostylar pharynx or gonads of M. manhattensis , incorporated 125 I into protein and free tyrosine in vitro . This iodinating activity was dependent upon a hydrogen peroxide-generating source and was inhibited either by boiling tissue or by the addition of methimazole. These properties suggest that the iodination process was mediated by a peroxidase. It is concluded that active binding of iodine occurs in both the endostyle and tunic of M. manhattensis . PB 125 I found in the extraendostylar pharynx and the gonads is suggested to have originated in other sites. Iodine binding in the endostyle appears to be mediated by an enzymatic mechanism similar to that of the vertebrate thyroid gland. Iodination in the tunic may be related both to quinone tanning and to the action of a peroxidase, either by two separate metabolic pathways or by a single process utilizing both quinones and a peroxidase.

Iodine metabolism in the red-spotted newt studied with radioactive tracer and by radioimmunoassay☆

Abstract Thyroidal function in the red-spotted newt was assessed both in 125 I tracer studies and in the measurement of glandular and circulating thyroxine (T 4 ) and triiodothyronine (T 3 ) by radioimmunoassay (RIA). Thyroidal uptake of 125 I was low, being 0.6% at 2 days and 3.6% at 14 days following injection of the tracer. A significant fraction of the injected dose continued to circulate as inorganic iodide at 7 and 14 days following injection, indicating a low rate of renal clearance for iodine. [ 125 I]T 4 represented only 6.2% of thyroidal 125 I at 2 days post-injection, but this figure had increased to 14% by 14 days, suggesting a slow but significant synthesis of hormone. By RIA the T 4 content in thyroids of animals freshly collected in the field was 3.42 ± 0.16 ug/mg protein, a level comparable to that found in beef thyroids. In contrast T 3 stores in the thyroid of the newt were only 9.1 ± 1.2 ng/mg protein. Although no [ 125 I]T 4 was detected in the serum of animals for as long as 21 days after injection, circulating thyroid hormones were detected by RIA at levels of 3.0 ± 0.3 ng T4hn1 and 0.28–0.72 ng T 3 /ml in freshly collected animals. The data suggest that T 4 in the serum may arise mainly from peripheral deiodination of T 4 . Large amounts of an iodoprotein with electrophoretic properties of thyroglobulin were found in the serum of newts. This protein contained 5% of the serum 125 I at 2 days postinjection and accounted for 30% of the serum radioactivity at 14 days. In captive newts, serum T 4 levels were three times greater and glandular T 4 levels approximately two times lower than those of freshly collected animals, suggesting that the newt thyroid responds to the stresses of captivity by accelerated release of thyroid hormone.

On the identity of the thyrotropic cell in the red-spotted newt

SummaryAlthough the pars distalis of the red-spotted newt has previously undergone extensive cytological examination, the identity of its thyrotropic cells has remained uncertain. From the present ultrastructural study, cells of type 3 (Masur, 1969) containing granules 150–180 nm in diameter are concluded to be the thyrotropes. Such cells were found to be present in the regions of the pars distalis where thyroidectomy cells arise after ablation of the thyroid gland. Cells of type 3 are probably identical with a population of cells containing granules which stain with silver methenamine indicating the presence of a glycoprotein such as thyroid stimulating hormone (TSH). Thyroidectomy cells containing a few residual granules 150–180 nm in diameter were occasionally found in partes distales from newts killed 3 or 7 days after ablation of the thyroid gland, and were abundant in pituitaries 21 days after thyroidectomy. Only cells of type 3 responded (by vacuolation of granules) when animals were immersed in water containing 10 μg/l of thyroxine. No cells of the pars distalis showed cytological change after administration of synthetic thyrotropic releasing hormone (TRH) giving additional support to the view that this hormone has no stimulatory role in amphibians.