Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.     

Mechanism of action of iodothyronine-5'-deiodinase.

Production of 3,3'-di-iodothyronine (3,3'-T2) from 3,3',5'-tri-iodothyronine (reverse T3, rT3) as catalysed by rat liver microsomal fraction was measured with a specific radioimmunoassay. The effect of the addition of 2-thiouracil and of varying concentrations of cofactor (dithiothreito) on the kinetic parameters of this reaction were studied. It was found that thiouracil is an uncompetitive inhibitor with respect to substrate and a competitive inhibitor with respect to cofactor. The effect of a decrease in the concentration of cofactor was similar to the effect of addition of thiouracil, i.e. a proportional decrease in Km and V. The results strongly suggest that enzymatic 5'-deiodination of iodothyronines follows a ping-pong mechanisms, which may be envisaged as a transiodination and the subsequent reduction of the iodo-enzyme complex by cofactor. The intermediate is probably a sulfenyl iodide form of the enzyme, which reacts with thiouracil to yield a mixed disulfide.     

pH-dependency of iodothyronine metabolism in isolated perfused rat liver.

1. Isolated livers from fed male rats were perfused for 2 h with T4 (L-thyroxine), T3 (L-3,3',5-tri-iodothyronine) or rT3 (L-3,3',5'-tri-iodothyronine) at different pH values (7.1--7.6) in a fully synthetic medium, whereby normal metabolic functions were maintained without addition of rat blood constituents or albumin. 2. T3 output into the medium and net T3 production reached a maximum at a pH of the medium of 7.2 and significantly decreased with alteration of the pH when livers were perfused with T4 as a substrate. 3. However, the net T4 and T3 uptake by the liver, as well as the hepatic T4 and T3 content after perfusion, were not dependent on the pH of the perfusion when livers were offered T4 or T3 as substrates respectively. 4. Determination of intracellular pH by the analysis of the distribution of the weak acid dimethyloxazolidinedione allows the conclusion that the pH optimum of iodothyronine 5'-deiodinase in the intact perfused liver corresponds to the maximum determined in vitro for the membrane-bound enzyme localized in the endoplasmic reticulum. 5. The rapid 5'-deiodination of rT3 to 3,3'-T2 (L-3,3'-di-iodothyronine), the fast disappearance of 3,3'-T2, and the fact that no net rT3 production from T4 could be detected, supports the hypothesis that in rat liver iodothyronine 5'-deiodinase activity seems to predominate over iodothyronine 5-deiodinase activity. 6. Thus the rat liver can be considered in normal physiological situations as an organ forming T3 from T4 and deiodinating rT3 originating from extrahepatic tissues, whereby the cellular iodothyronine 5'-deiodination rate is controlled by the intracellular pH.     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Kinetics of enzymic reductive deiodination of iodothyronines. Effect of pH.

5'-Deiodination of thyroxine (yielding 3,3',5-tri-iodothyronine; reaction I) and of 3,3',5'-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction II) and 5-deiodination of thyroxine (yielding 3,3',5'-tri-iodothyronine; reaction III) and of 3,3',5-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction IV) as catalysed by rat liver microsomal fraction were studied at pH 6.5, 7.2 and 8.0 It was found that: (1) the Km of reaction I was relatively independent of pH (approx. 3 microM), whereas V was highest at pH 6.5 (63 pmol of 3,3',5-tri-iodothyronine/min per mg of protein); (2) the Km of reaction II was lowest at pH 6.5 (0.035 microM), but V was highest at pH 8.0 (829 pmol of 3,3'-di-iodothyronine/min per mg of protein); (3) thyroxine inhibited reaction II competitively; Ki values were identical at pH 6.5 and 8.0 (1 microM); (4) for both reactions III and IV Km was lowest and V was highest at pH 8.0. The results are compatible with the view that reactions I and II are mediated by a single enzyme (iodothyronine 5'-deiodinase) and that reactions III and IV are catalysed by a second enzyme (iodothyronine 5-deiodinase).     

Effect of ethanol and linoleic acid on changes in biliary excretion of iodothyronines possibly related to thyroxine deiodination in rat liver

Groups of male rats weighing about 350 g were inserted polyethylene tubings into bile duct and femoral vein under pentobarbital anesthesia. Several iodothyronines (i.e. T4, T3, rT3, 3,5-T2, 3,3'-T2 and 3',5'-T2) were estimated in 2-hr portions of bile with the aid of specific radioimmunoassay. After the infusion of ethanol (0.3 ml/hr/rat for 4 hr) an increase of biliary excretion of rT3 and a decrease of 3,5-T2 was found as compared to controls. When 5 mg linoleic acid was added to 1.2 ml ethanol, the increase of rT3 was significantly higher than that after ethanol only and, in addition, significant increase of 3',5'-T2 excretion was found. It was concluded that both ethanol and unsaturated fatty acids may inhibit 5'-monodeiodination in the liver and that unsaturated nonesterified fatty acids may exert such effect even when administered intravenously without underlying metabolic disorders.     

Existence of extrathyroidal conversion inhibitor (IEC) in starved animals and its influence on thyroid hormones deiodination in liver and kidney in vitro

To find out whether an inhibitor of extrathyroidal conversion of iodothyronines is present in sera of starved animals, pig liver and kidney homogenates were incubated with T4, T3 or rT3 and dithiotreitol in the presence of evaporated diethyl ether extracts of sera obtained from fed and starved (1-12 days) rabbits. Sera extracts of short-term (1-4 days) starved rabbits caused a significant inhibition of T4 to T3 conversion (54% on day 3) and T4 to rT3 deiodination (52% on day 2) in liver homogenates. Extracts of sera from long-term (8 and 12 days) starved animals diminished only liver T4 to T3 conversion on day 8 and had no influence on liver T4 to rT3 conversion. 5'-deiodination of rT3 (to 3,3'-T2) in liver was gradually decreased by extracts of sera from animals starved during 2-12 days. Liver rT3-5-deiodination (to 3',5'-T2) was significantly impaired on day 4 and totally depressed by long-term starvation. In vitro T3 to 3,3'-T2 conversion in liver was markedly (59-103%) increased by ether extracts of sera from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term nor from long-term fasted rabbits but T4-5-deiodination (to rT3) was reduced by sera extracts of short-term fasted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential deiodination of thyroxine in rat liver homogenate.

Rat liver homogenate was incubated at 37 degrees C with thyroxine, 3,3',5-tri-iodothyronine, 3,3',5'-tri-iodothyronine or 3,3'-di-iodothyronine. The degradation or accumulation of these compounds was measured by specific radioimmunoassays. (1) Production of 3,3',5-tri-iodothyronine from thyroxine was highest at pH 6.0--6.5 and was markedly stimulated by the addition of dithiothreitol and effectively inhibited in the presence of 6-propyl-2-thiouracil. (2) Accumulation of 3,3',5'-tri-iodothyronine on incubation of thyroxine with homogenate was only observed above pH 8.5. Otherwise the product was converted into 3,3'-di-iodothyronine too rapidly to allow its measurement. By measuring 3,3'-di-iodothyronine it was deduced that 5-deiodination of thyroxine was most effective at approx. pH 8.0. Dithiothreitol powerfully stimulated this reaction and 6-propyl-2-thiouracil strongly inhibited. (3) Monodeiodination of the tyrosine ring of 3,3',5-tri-iodothyronine was the slowest reaction, was optimal at pH 8.0 and was less affected by dithiothreitol and 6-propyl-2-thiouracil than the above reactions. (4) 5'-Deiodination of 3,3',5'-tri-iodothyronine was extremely rapid, with a pH optimum probably at about 6.5. Owing to the high reaction rate under the conditions used it was not possible to assess the effects of dithiothreitol and 6-propyl-2-thiouracil.     

Triiodothyronamine--a beta-adrenergic metabolite of triiodothyronine?

Triiodothyronamine (Triam) is a potential metabolite of triidothyronine (T3), resulting from decarboxylation of the side-chain. In an attempt to elucidate the physiological properties of Triam we have investigated the binding of Triam to beta-adrenergic receptors, using turkey-erythrocytes and performing binding studies with ( (-)(3H)-dihydroalprenolol) ( (-)(3H)-DHA) as a specific beta-adrenergic ligand. The inhibition constant Ki for Triam was determined as 5 X 10(-6) M, compared to dopamine (Ki = 1,3 X 10(-2) M), norepinephrine (Ki = 3 X 10(-4) M), epinephrine (Ki = 5 X 10(-5) M) and isoproterenol (Ki = 3 X 10(-6) M). The inhibition of ( (-)(3H)-DHA)-binding by Triam was further compared with other iodothyronines thyroxine (T4), T3, 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2). It is concluded that Triam binds to beta-adrenergic receptors like naturally occurring amines but different from typical circulating iodothyronines.     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

The substrate specificity, tissue specificity and regulation of the 5' deiodination systems in rat liver and kidney tissues

Studies were carried out to compare the 5' deiodination reactions of thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) in rat liver and kidney homogenates. The 5'-deiodinase activity was assayed by the 3, 5, 3'-triiodothyronine (T3) produced from T4 or by the 125I-iodide released from 125I-rT3. The two 5' deiodination reactions had similar ranges of optimal pH, incubation temperature, and apparent Km, T4 1.1 and rT3 1.3 microM. However, the apparent Vmax values for T4 and rT3 deiodination reactions were 0.9 and 220 pmol/mg protein/min, respectively. Both reactions were stimulated by thiol reagent but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil (PTU) on the 5' deiodination of rT3 was 50 times as great as that of T4. Only the 5' deiodination of rT3 was inhibited by low concentrations of calcium and magnesium. The 5' deiodination reactions in the liver and kidney tissues showed very similar substrate specificity. However, only the hepatic deiodinase activity was reduced to 60-65% of the control value after fasting, whereas the renal 5'-deiodinase activity was unaffected or even enhanced by fasting up to 72 hours. The results showed the existence of a diverse and complex 5' deiodination system in the rat tissues which is comprised of multiple similar but distinct 5'-deiodinase enzymes with respect to their substrate specificity, tissue specificity and regulation.     

Substrate specificity of iodothyronine 5'-deiodinase in rat liver homogenates and its requirements of divalent cations in vitro

Studies were carried out to compare the 5'-deiodination reactions of thyroxine (T4) and 3,3'-5'-triiodothyronine (rT3) in 2.5% rat liver homogenates. The 5'-deiodinase activity was assayed by the 3,5,3'-triiodothyronine (T3) produced from T4 or by 125I-rT3. Under our experimental conditions, the two 5'-monodeiodination reactions resulted in similar apparent KMs: 1.5 microM for T4 and 1.1 microM for rT3. However, the apparent Vmax values of T4 and rT3 deiodination reactions were, respectively, 0.91 and 222 pmol/mg protein/min. Both reactions were stimulated by thiol reagents but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil on the 5'-deiodination of rT3 was at least 50 fold greater than that of T4. The divalent ion requirement of the deiodination system was tested with CaCl2, MgCl2, and ZnCl2 at a range of concentrations. Zinc ion appeared to be a potent inhibitor in both T4 and rT3 deiodination systems. Only the 5'-deiodination of rT3 was inhibited slightly by low concentrations of calcium and magnesium ions. Our results suggest that based on their apparently distinct regulation mechanisms, the 5'-monodiodination of T4 and rT3 in rat liver homogenates is likely mediated by more than one enzyme, despite the similarity of observed KMs.     

Effect of 3,3',5' triiodo-L-Thyronine (rT3) on the serum concentration of thyroid hormones in rats

50, 100 or 150 micrograms/100 g body weight/day of very pure 3,3',5' triiodo-L-thyronine (rT3), obtained from a new synthetic method, was intraperitoneally administered in male Wistar rats for 5 weeks. Serum total thyroxine (T4), free thyroxine (FT4) and total 3,5,3' triiodo-L-thyronine (T3) concentrations were increased with all the doses of rT3. Free T3 (FT3) was also but non-significantly elevated. Different assumptions are put forward in order to explain this rT3 effect.     

Effects of streptozotocin-induced diabetes mellitus on hypothalamic-pituitary-thyroid axis in rats

The effects of streptozotocin-induced diabetes mellitus on the hypothalamic-pituitary-thyroid axis in rats were studied. Streptozotocin (60 mg/kg) was injected ip. Rats were decapitated at two and four weeks after the streptozotocin treatment. Thyrotropin releasing hormone (TRH), thyrotropin (TSH), thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) were measured by means of the specific radioimmunoassay for each. Immunoreactive TRH (ir-TRH) contents in the hypothalamus significantly decreased at four weeks (p less than 0.02). Basal TSH levels in plasma significantly decreased (p less than 0.005, p less than 0.001), and plasma ir-TRH and TSH responses to cold were significantly inhibited after the streptozotocin treatment (p less than 0.001). The plasma TSH response to TRH was decreased, but not significantly. The plasma T4 and T3 levels fell significantly. RT3 did not change throughout the experiment. 3,3'-T2 levels in plasma fell significantly, whereas 3',5'-T2 increased. Blood glucose levels rose significantly after streptozotocin treatment, but insulin treatment led to partial restoration. The findings suggest that streptozotocin-induced diabetes mellitus affects various sites of the hypothalamic-pituitary-thyroid axis in rats.     

Thyroid status in the obese syndrome of rats

The thyroid function was explored by comparing serum total and free iodothyronine levels in young male genetically obese Zucker rats and in their lean littermates, aged from 6 to 8 weeks old. Total and free thyroxine (T4) and 3,5,3'triiodothyronine (T3) levels were significantly decreased in obese rat serum while total 3,3',5'-triiodothyronine (rT3) remained constant. Radioactive T4 half life is slower in the plasma of obese rats. Peripheral synthesis of T3 from deiodination of T4 is also decreased in obese rat liver homogenate. These modifications produce changes in liver mitochondria oxidative phosphorylation and in marker enzyme activity, which are usually associated with hypothyroidism and hypothalamic disturbances. Genetic obesity probably involves activation of peripheral deiodination of T4 to rT3 which induces biochemical and metabolic changes.     

Placental outer and inner ring monodeiodination of thyroxine and triiodothyronines in the rabbit

Both inner- and outer-ring iodothyronines deiodinating activity was found in homogenates of rabbit placentas. The T4 to rT3 and T3 to 3,3'-T2 deiodinating activity was already high on day 10 before delivery but decreased being about 7 times lowered on day 5. Once the T3 to 3,3'-T2 monodeiodination reached a low and a relatively steady level, the outer ring deiodination of T4 begun, reaching a peak value at about day 3 before term and then fell again. The fetal serum thyroid hormones levels were low, showing no significant variability during the period of observation. The results suggested that in the rabbit, representing animals in which the thyroid gland activity begins early in fetal life, there are two distinct phases of the placental monodeiodinating activity. The first is characterized by a high inner-ring deiodinating activity (yielding rT3) and is followed by the second phase with a high outer-ring deiodinating activity (yielding T3) declining just before term.     

Characterization of rat iodothyronine sulfotransferases

Sulfation appears to be an important pathway for the reversible inactivation of thyroid hormone during fetal development. The rat is an often used animal model to study the regulation of fetal thyroid hormone status. The present study was done to determine which sulfotransferases (SULTs) are important for iodothyronine sulfation in the rat, using radioactive T4, T3, rT3, and 3,3'-T2 as substrates, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as cofactor, and rat liver, kidney and brain cytosol, and recombinant rat SULT1A1, -1B1, -1C1, -1E1, -2A1, -2A2, and -2A3 as enzymes. Recombinant rat SULT1A1, -1E1, -2A1, -2A2, and -2A3 failed to catalyze iodothyronine sulfation. For all tissue SULTs and for rSULT1B1 and rSULT1C1, 3,3'-T2 was by far the preferred substrate. Apparent Km values for 3,3'-T2 amounted to 1.9 microM in male liver, 4.4 microM in female liver, 0.76 microM in male kidney, 0.23 microM in male brain, 7.7 microM for SULT1B1, and 0.62 microM for SULT1C1, whereas apparent Km values for PAPS showed less variation (2.0-6.9 microM). Sulfation of 3,3'-T2 was inhibited dose dependently by other iodothyronines, with similar structure-activity relationships for most enzymes except for the SULT activity in rat brain. The apparent Km values of 3,3'-T2 in liver cytosol were between those determined for SULT1B1 and -1C1, supporting the importance of these enzymes for the sulfation of iodothyronines in rat liver, with a greater contribution of SULT1C1 in male than in female rat liver. The results further suggest that rSULT1C1 also contributes to iodothyronine sulfation in rat kidney, whereas other, yet-unidentified forms appear more important for the sulfation of thyroid hormone in rat brain.     

Conversion of thyroxine into 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine in the young pig

Estimates have been made of the amounts of 3,5,3'-triiodothyrone (T3) and 3,3',5'-triiodothyronine (rT3) derived from peripheral deiodination of thyroxine (T4) in young pigs. Two methods were used. The first depended on the assumption that deiodination occurs at the same rate in normal animals and in thyroidectomized animals on T4 replacement therapy. The second on the assumption that T3 and rT3 are secreted in the same proportions as they occur in thyroglobulin. The first method arguably gives the better estimate which is that 87% of circulating T4 is monodeiodinated to T3 and rT3. Peripheral conversion accounts for 76 and 69% of the circulating T3 and rT3, respectively.     

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5''-deiodinase.

The properties and kinetic characteristics of a non-GSH NADPH-dependent cofactor system activating rat hepatic and renal 5'-deiodinase (5'-DI), which we have previously demonstrated with partially purified cytosol Fractions A and B [Sawada, Hummel & Walfish (1986) Biochem. J. 234, 391-398], were examined further. Although microsomal fractions prepared from either rat liver or kidneys could be activated by crude cytosol Fractions A and B from those tissues as well as from rat brain and heart, a homologous hepatic or renal system was the most potent in producing 5'-deiodination of reverse tri-iodothyronine (rT3). At nanomolar concentrations both rT3 and thyroxine (T4) were deiodinated but with a much greater substrate preference for rT3 than for T4. However, at micromolar concentrations of these substrates no activation of 5'-DI could be detected. In this deiodinative system, T4 and tri-iodothyronine (T3) competitively inhibited 5'-deiodination of rT3. Dicoumarol, iopanoate, arsenite and diamide were also inhibitory to the activation of hepatic or renal 5'-deiodination by this cofactor system. Purification of cofactor components in hepatic crude cytosolic Fractions A and B to near homogeneity, as assessed by their enzymic and physical properties, indicated that these co-purified with and were therefore identical with thioredoxin reductase and thioredoxin respectively, and accounted almost entirely for the observed activation of rT3 5'-DI. When highly purified liver cytosolic thioredoxin reductase and thioredoxin were utilized to determine the kinetic characteristics of the reaction, evidence for a sequential mechanism operative at nanomolar but not micromolar concentrations of rT3 and T4 was obtained. The Km for rT3 was 1.4 nM. Inhibition by 6-n-propyl-2-thiouracil (Ki 6.7 microM) was competitive with respect to thioredoxin and non-competitive with respect to rT3, whereas inhibition by T4 (Ki 1.3 microM) was competitive. Since rT3 is a potent inhibitor of T4 5'-deiodination, this thioredoxin system activating deiodination of rT3 may play an important role in regulating the rate of intracellular production of T3 from T4.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.