[Studies on the metabolism of L-diiodotyrosine. 2. Metabolism of I-131-labelled L-diiodotyrosine in various thyroid diseases].
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Diiodotyrosine↗
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[Studies on the metabolism of L-diiodotyrosine. 3. Defectve deiodination of I-131-labelled L-diiodotyrosine in patients with simple goiter].
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Diiodotyrosine↗
Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.
Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.
Animals↗
Interaction of lactoperoxidase with thiols and diiodotyrosine.
Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.
Cysteine↗
Photoaffinity labeling of lysosomal membrane proteins with [125I]diiodotyrosine, a system h ligand.
Percoll-purified rat thyroid FRTL-5 cell lysosomes were photoaffinity-labeled with [125I]diiodotyrosine to identify proteins which bind diiodotyrosine, a ligand for lysosomal transport system h. SDS-PAGE and autoradiography of these membranes showed specific labeling of a 70-kDa protein and weak labeling of three smaller proteins. [125I]Diiodotyrosine photolabeling of the 70-kDa protein was specifically competed against by ligands of lysosomal transport system h ligands. The 70-kDa protein was photolabeled more strongly in lysosomal membranes isolated from thyrotropin-stimulated cells when compared with those grown in the absence of thyrotropin, consistent with previous demonstrations that thyrotropin stimulates system h transport. The 70-kDa protein may represent some portion of the system h carrier protein.
Affinity Labels↗
Effects of small doses of bovine TSH on serum levels of free and total thyroid hormones, their degradation products, and diiodotyrosine.
Bovine TSH was administered iv to 10 normal volunteers in doses of 2.5, 7.5, 15 and 30 mU/kg. Brisk elevations of serum diiodotyrosine occurred already after the smallest dose (mean, +183%) while larger doses had only slight additional effects. T3 rose much higher than T4 (+71% compared to +23% after 15 mU bTSH/kg), and free thyroid hormones exhibited changes similar to total T3 and total T4. The mean absolute increase in serum fT3 ranged from 2.03 to 9.04 pmol/l and proved to be an easily measurable parameter for the TSH effect. Dose-response effects were seen for the increase of fT4, fT3 and T3. TBG and rT3 did not change but the degradation product 3,3'-T2 showed large increments of serum levels. There was no correlation between the response of T3 and T4, fT3 and fT4, or diiodotyrosine and any of the other parameters of thyroid function. The interindividual differences in the magnitude of thyroid hormone response to TSH were considerable, and there was no relationship between this response and thyroid volume by ultrasound. We conclude that direct stimulation of the thyroid gland with bTSH in small doses leads to consistent increases of thyroid hormones, especially T3 and fT3, that the response varies between individuals, and that the precursor diiodotyrosine is released together with thyroid hormones.
Adult↗
Thyroid hormone contamination in crystalline diiodotyrosine.
Crystalline preparations of diiodotyrosine contain trace quantities of thyroxine that can be detected by several competitive radioassays and by chromatography on Sephadex. Spontaneous coupling of diiodotyrosine to thyroxine during iodination of tyrosine and coprecipitation of hormone with diiodotyrosine crystals are likely explanations for the trace contamination. We discuss the significance of these findings in relation to studies on the biosynthesis of thyroxine.
Diiodotyrosine↗
Response of total and 'free' thyroid hormones and diiodotyrosine to bovine TSH in subclinical hypothyroidism.
Thirty-three patients with Addison's disease were studied. Twenty-two had idiopathic Addison's disease; within this group, 14 patients had clinical or subclinical hypothyroidism, and 16 had increased titres of thyroid autoantibodies. Five patients had tuberculous, and eight had unclassifiable Addison's disease; only one patient in the latter group had evidence of thyroid autoimmunity. A stimulation test with 15 mU bTSH/kg was performed in three patients with Schmidt's syndrome (coexisting Addison's disease and manifest primary hypothyroidism), 15 patients with either subclinical hypothyroidism or increased titres of thyroid autoantibodies, 10 patients without thyroid involvement, and 10 normal controls. There was no detectable increase of 'free' and total thyroid hormones in Schmidt's syndrome. The mean increases after 3-4 h of T4, fT4, T3 and fT3 were 22, 35, 63 and 66%, respectively, in patients without thyroid involvement, and 13, 24, 46 and 45% in patients with subclinical hypothyroidism. 'Free' but not total thyroid hormones rose significantly (P less than 0.01) higher in patients without signs of thyroid involvement than in patients with subclinical hypothyroidism and/or thyroid autoantibodies. Thyroid hormone response to bTSH in Addison's disease with apparently healthy thyroid glands was not different from normal controls. Serum diiodotyrosine rose in all groups except in hypothyroidism; hypothyroid patients had, however, basal levels well within the normal range. Thus, thyroid hormone synthesis appears to be blocked at a point distal to diiodotyrosine formation in this particular situation. These results support the assumption that TSH elevation in idiopathic Addison's disease is due to coexisting thyroid autoimmunity and that it reflects incipient thyroid failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Addison Disease↗
One- and two-electron oxidations of tyrosine, monoiodotyrosine, and diiodotyrosine catalyzed by hog thyroid peroxidase.
Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). In the steady state of oxidations of L- and D-tyrosines, N-acetyltyrosinamide, and monoiodotyrosine, thyroid peroxidase existed in the form of Compound I, the primary catalytic intermediate of peroxidase in its reaction with H2O2. Kinetic results led us to conclude that thyroid peroxidase catalyzes two-electron oxidations of these molecules. In the steady state of oxidation of diiodotyrosine, on the other hand, the enzyme was found in the form of compound II at pH 7.4, but in the form of compound I at pH 5.5. The result implies that the mechanism of diiodotyrosine oxidation varied from a one-electron to a two-electron type as the pH decreased. The selection of mechanisms of oxidation appears to be peculiar to thyroid peroxidase; horseradish peroxidase and lactoperoxidase catalyzed only one-electron oxidations of these five donor molecules. Rate constants for rate-limiting steps in the reactions of these donor molecules with the three peroxidases were measured by overall kinetic and stopped flow kinetic methods.
Animals↗
Gas-liquid chromatographic determination of mono- and diiodotyrosines in serum.
A sensitivie, reliable gas-chromatographic assay for monoiodotyrosine and diiodotyrosine in human serum is reported. The oxazolidinone-heptafluorobutyric anhydride derivatives allow the quantitation of both compounds in the linear range of 0.2 to 7.6 mg/L of serum. Analytical recovery averaged 88%, and mean accuracy and within-run precision were 98 and 2%, respectively. Concentrations of monoiodotyrosine in serum as low as 20 microgram/L and of diiodotyrosine as low as 100 microgram/L can be detected. Normal serum contains no detectable concentration of either compound, but the method is applicable as a diagnostic tool in the early prediction of thyroid disease. Both compounds were detected in the serum of a hypothyroid subject whose normal thyroid hormone concentrations were being maintained by therapy with desiccated thyroid extract.
Butyrates↗
3,5-Diiodotyrosine excretion in human urine in individuals of differing thyroidal status.
The urinary excretion of 3,5-diiodotyrosine was determined in euthyroid, hypothyroid and hyperthyroid individuals using a sensitive gas chromatographic mass spectrometric assay. This involved a multi-step extraction of the amino acid from urine (mean efficiency 28 +/- 5.7%) then conversion to the N,O-diheptafluorobutyryl methyl ester. The fragmentation of the derivative is discussed. Although the mean excretions in the two pathological states were significantly different from that of euthyroid individuals (P less than 0.01 in both instances) there was considerable overlap with the normal range.
Diiodotyrosine↗
[In vitro study of deiodination of L-diiodotyrosine and L-thyroxine in liver of dietetically fat rats].
Beginning with the fifth week of life, male Wistar rats were fed a diet containing, respectively, 3 and 50 per cent fat (corresponding to, respectively, 7 and 75 per cent fat of total calories). The differences in body weight between the two groups were 64 per cent for 10-week-old rats, and 71 per cent for 6-month-old rats. For these animals, the extent of the enzymatic deiodination of L-diiodotyrosine and L-thyroxin was determined in liver homogenate supernatants. In the 10-week-old rats as well as in the 6-month-old rats on high-fat diet, the deiodination of L-di-iodotyrosine was significantly lower than in the animals on low-fat diet, whereas the diodination of L-thyroxin was significantly higher. It is concluded that the fat content of the diet exerts an inhibiting effect on the deiodination of L-di-iodotyrosine and an enhancing effect on the deiodination of L-thyroxin.
Age Factors↗
B1-3,5-diiodotyrosine insulin: a valid tracer for insulin.
Insulin, specifically substituted at the PheB1 position with 3,5-diiodotyrosine, has been tested in several biological and immunological systems. Immunoreactivity was assessed using antisera specific for different parts of the insulin molecule. Biological activity in vitro was estimated on isolated rat fat cells. In vivo bioactivity (hypoglycaemia) and metabolism (metabolic and urinary clearance rates, half-life, apparent distribution space) were measured by infusion of the material into greyhounds. The results indicated that this B1-labelled insulin preparation was biologically fully active and, unlike randomly labelled preparations of iodoinsulin, was metabolised with kinetics indistinguishable from those of the unlabelled hormone. We suggest that this material is a valid tracer for insulin, fulfilling the criteria of high specific activity and biological identity to the native hormone.
Adipose Tissue↗
Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone.
Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower Vmax than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.
Adenylyl Cyclases↗
Binding of 3,5-diiodotyrosine to serum proteins.
The reversible binding of 3,5-diiodotyrosine (DIT) to human and bovine serum protein and to purified human serum prealbumin and human and bovine albumin has been studied by equilibrium dialysis. Maximum binding occurred at pH 8.6-9.0. Human serum bound DIT less than did bovine serum. Adult ox and fetal calf sera showed similar binding. The main DIT-binding protein of human serum was prealbumin. It showed a single affinity site with a Ka of 0.85 X 10(6) M-1 at pH 8.6 and 0.40 X 10(6) M-1 at pH 7.4. The affinity constant of serum albumin for DIT was 2.8 X 10(3) M-1 at pH 8.6. The elevated binding of DIT to bovine serum is essentially due to albumin whose affinity constant for DIT is 16-times higher than that of human serum albumin. Fetuin was not responsible for any noticeable DIT binding in fetal calf serum.
Binding Sites↗
Electron microscopic localization and hormonal (diiodotyrosine) induction of adenylate cyclase in the Tetrahymena.
Normal Tetrahymena cells exhibited adenylate cyclase activity exclusively in association with pinocytotic vesicles, whereas those treated with diiodotyrosine (T2) for growth stimulation showed it in association with the cell membrane, and intracellularly inside many dense bodies. It appears that hormonally activable adenylate cyclase is an inherent component of the Tetrahymena.
Adenylyl Cyclases↗
Repair of semi-oxidized 3,5-diiodotyrosine: radiation chemical studies in the presence of oxygen, ascorbate and superoxide anion.
Reactions of semi-oxidized radicals derived from 3,5-diiodotyrosine (I2TyOH, a thyroid hormone precursor) have been studied using radiation chemical techniques. In buffered, aqueous medium at room temperature, molecular oxygen reactivity towards the phenoxyl radical (I2TyO.) is low, the average bimolecular rate constant, k being 1.7 +/- 0.22 x 10(6) dm3 mol-1s-1. On the other hand, superoxide anion (O2-) reactivity towards I2TyO. is close to the diffusion controlled limit, the k being 5 +/- 1 x 10(9) dm3 mol-1s-1. The major reaction channel in this case (approximately 60%) leads to the reformation of the parent compound by one-electron transfer. Under similar experimental conditions, ascorbate (As-) completely reduces I2TyO. to the parent compound with k = 3 +/- 0.5 x 10(9) and < or = 1 x 10(9) dm3 mol-1s-1 at pH 7.4 and 12 respectively. The propensity of these reactions are not dependent on the primary .OH/.O- or secondary N3. radicals used. These results suggest that the superoxide anion may actively interact at the cellular level, in the Thyroid during the course of I2TyOH oxidation, and the observed in vitro reaction mechanism implies its participation in a new role.
Ascorbic Acid↗
3,5-Diiodotyrosine and thyronine in the urine of patients with chronic renal disease.
Urinary 3,5-diiodotyrosine (DIT) and thyronine (T0) excretion was investigated in 18 patients with chronic renal disease. In accord with previous findings serum T4 and thyroid hormone binding proteins measured in 17 patients were in the low or normal range. Urinary albumin excretion was elevated in all 18 and T4 binding prealbumin (TBPA) in 15 of the 18. Urinary T0 excretion measured in 12 patients was also significantly lower than normal (mean +/- SD 4.4 +/- 2.6 vs 15.8 +/- 5.8 nmol/24 h renal vs normal 2 P less than 0.001). In contrast urinary DIT excretion was significantly elevated in renal patients compared with normal subjects (2.0 +/- 1.5 vs 0.75 +/- 0.41 nmol/24 h, respectively). Possible sources of the increased DIT are discussed.
Adult↗