Thyroid hormone deiodination in tissues of American plaice, Hippoglossoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126

We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels to an intraperitoneal injection (5-500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3'-4,4'-tetrachlorobiphenyl) or 126 (3,3',4,4',5-pentachlorobiphenyl). T4 and 3,3'5'-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent Km values of 0.6-4 nM) fell in the same rage as those of low-Km deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25 degrees C but uniformly low over the natural range of 0-9 degrees C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P < 0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.     

Intra- and extra-cellular sources of T3 binding to putative thyroid hormone receptors in liver, kidney, and gill nuclei of immature rainbow trout, Oncorhynchus mykiss.

The sources of extracellular and intracellular 3,5,3'-triiodo-L-thyronine (T3) binding to putative thyroid hormone receptors in liver, kidney, and gill nuclei were determined in vivo for immature rainbow trout at 12 degrees C. Both [131I]T3 and [125I]T4 were injected intraperitoneally, the plasma and tissues were examined at isotopic equilibrium at 20 h, and the proportions of intracellular [125I]T3 and extracellular [131I]T3 saturably bound in the nucleus were determined. Comparable total amounts of T3 were saturably bound in the nuclei of liver (7.2), kidney (8.0), and gill (9.7 moles x 10(-13) .mg DNA-1), but the percentage of nuclear T3 generated within the target cell was greater for gill (76%) than for liver (50%) and kidney (28%). Both gill and liver possess a low Km T4 5'monodeiodinase which could be responsible for the high proportion of the nuclear T3 generated within those tissues.     

Hepatic 5'-deiodinase activity of Japanese quail using reverse-T3 as substrate: assay validation, characterization, and developmental studies

Using rT3 as substrate, an in vitro 5'D assay was validated for use with liver tissue from adult Japanese quail, by defining conditions under which activity is proportional to enzyme (protein) concentration and is linear with incubation time. Activity was measured as the release of 125I from labeled rT3. Using validated assay conditions we found the following 5'D characteristics: maximal activity from 10 to 50 mM dithiothreitol (cofactor), an apparent Km of 0.52 microM rT3, pH optimum of 7.6-8.5, complete inhibition by 1 mM propylthiouracil and by 1.0 mM iopanoic acid, and substrate "preference" of rT3 greater than T4 greater than T3. Based on these characterizations the quail hepatic 5'D activity is like the Type I 5'D activity found in mammalian liver and kidney and embryonic chicken liver. To determine how previous unvalidated assays, that used high tissue and relatively low substrate (T4) concentrations, influenced 5'D studies we reevaluated 5'D development using an assay validated for each developmental stage with rT3 as substrate. We found extreme quantitative differences in the activities measured and in the proportional relationships between stages, and only limited qualitative similarity in the pattern of 5'D development when unvalidated T4 assay results were compared with validated rT3 assay results. Our data in this paper show good correspondence between whole liver 5'D activity per unit body weight and plasma T3/T4 ratios for the developmental stages sampled.     

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.     

Characterization of type-II thyroxine 5'-deiodinase activity in rat Harderian gland

Thyroxine 5'-deiodinase activity was studied in male rat Harderian gland homogenates. The reaction rate was proportional to the tissue content in the homogenate and dependent on pH, with an optimum pH of 7.0, and temperature, between 4-37 degrees C. 5'-deiodinase activity was increased by dithiothreitol (DTT) in a dose-dependent manner, and inhibited moderately by propylthiouracil (PTU) and strongly by iopanoic acid (IA). Thyroidectomy enhanced the enzymatic activity (30-fold above the control value) but this increase is totally prevented by the in vivo iopanoic acid treatment. Thyroxine 5'-deiodinase activity was also dependent on T4 concentration (Km = 3.3 nM; Vmax = 10 fmol 125I-released/mg protein/h) and exhibited a nyctohemeral rhythmicity with a maximal activity at 03.00 h (4-fold above basal values) and minimal activity between 12.00-21.00 h.     

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.     

Characterization of glutamine uptake in mouse two-cell embryos and blastocysts.

Mouse two-cell embryos and blastocysts take up [3H]glutamine in vitro at a constant rate for at least 15 min, depending on the concentration of glutamine and developmental stage of the embryo. Uptake by two-cell embryos can be resolved into two saturable components. The major contributing system is Na+ independent, inhibited by alanine, methionine, 2-amino-2-norbornanecarboxylic acid (BCH) or leucine and has a Km of 3856 +/- 672 mumols l-1 and Vmax of 436 +/- 58 fmol per embryo per 10 min. These features are characteristics of the ubiquitous system L transporter. The second component is Na+ dependent with Km of 1064 +/- 914 mumols l-1 and Vmax 107 +/- 47 fmol per embryo per 10 min. Similar Vmax and inhibition of this component by glycine suggest a low reactivity with the gly-system. Blastocyst uptake of glutamine is mainly by a Na(+)-dependent saturable mechanism with Km of 524 +/- 75 mumols l-1 and Vmax of 1264 +/- 101 fmol per embryo per 10 min which is inhibited by alanine, isoleucine, leucine and BCH, features characteristic of the system B0,+. The increase in uptake capacity as a consequence of the appearance of the system B0,+ may be related to increased metabolic requirements for glutamine, in the rapidly expanding blastocyst.     

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.     

Bioenergetic impact of tissue-specific regulation of iodothyronine deiodinases during nutritional imbalance

The regulation of energy homeostasis by thyroid hormones is unquestionable, and iodothyronine deiodinases are enzymes involved in the metabolic activation or inactivation of these hormones at the cellular level. T3 is produced through the outer ring deiodination of the prohormone T4, which is catalyzed by types 1 and 2 iodothyronine deiodinases, D1 and D2. Conversely, type 3 iodothyronine deiodinase (D3) catalyzes the inner ring deiodination, leading to the inactivation of T4 into reverse triiodothyronine (rT3). Leptin acts as an important modulator of central and peripheral iodothyronine deiodinases, thus regulating cellular availability of T3. Decreased serum leptin during negative energy balance is involved in the down regulation of liver and kidney D1 and BAT D2 activities. Moreover, in high fat diet induced obesity, instead of increased serum T₃ and T₄ secondary to higher circulating leptin and thyrotropin levels, elevated serum rT3 is found, a mechanism that might impair the further increase in oxygen consumption.     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.     

Acute cold exposure,leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of (125)I from (125)I-reverse triiodothyronine (rT(3)) under different assay conditions. Rats exposed to 4 degrees C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching approximately 40% at 2 h (P < 0.05) despite increased circulating TSH (P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 microg/100 g body wt sc) induced increased thyroid and liver D1 (P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 microg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.     

Simultaneous observations of iodothyronine content in the thyroid gland, serum and thyroxine 5'- and 5-monodeiodinase activity in liver, during the neonatal period of the pig

Serum T4 and rT3 were high at about 4-12 h after birth, then they decreased to a nadir on day 3 (rT3) and day 7 (T4). Serum T3 concentration fell immediately after birth but then increased to a relatively stable level during the next 2-6 weeks, then fell after weaning. Reciprocal concentration profiles of T4, T3 and rT3 in the thyroid were found. The thyroidal iodothyronine content increased significantly after weaning. In the liver, 5'-monodeiodinating activity, low after birth, rose until day 3 and then decreased concomitantly with T3 in serum. The 5-monodeiodinating activity, high at birth, fell to a nadir at about 3 weeks. No changes in 5- and 5'-deiodinase activity after 3 weeks were observed. Opposite to the variations in absolute content, the iodothyronine relative proportion in thyroid tissue was practically unchanged until weaning time (6 weeks), when they rose. Serum T3/T4 and rT3/T4 ratios increased with age until weaning. The post-weaned pigs had T3/T4 and rT3/T4 ratios about two times smaller than 6-weeks-old pigs. Serum rT3/T3, high after birth, decreased with age. Summarizing, the results indicate that neither changes in the thyroid iodothyronine content nor in the liver T4-monodeiodinating activity can solely account for variations in serum TH during the early neonatal period in the pig. It is suggested that the rapid variations in serum TH levels can reflect changes in the thyroidal secretory activity in preferential T3 secretion and/or blood disappearance rates.     

Effects of thyroid hormones and reverse-triiodothyronine (rT3) pretreatment on beta-adrenoreceptors in the rat heart

Effects of thyroxine (T4), triiodothyronine (T3) or reverse-triiodothyronine (rT3) in vivo pretreatment or the effect of hypothyroidism on properties of beta-adrenoreceptors in the rat heart were investigated. beta-adrenergic antagonist hydroxybenzylpindolol (HYP125I) was used to estimate the maximal binding capacity (MBC) and the affinity (KD) of beta-adrenoreceptors. Both T4 and T3 in dose and time dependent manner increased MBC value but only slightly and insignificantly affected affinity of the beta-adrenoreceptor. The effect of T3 on MBC was higher than that of an equal dose of T4, but the latter effect persisted longer than that of T3. Hypothyroidism decreased MBC value, but increased the affinity of beta-adrenoreceptors. Pretreatment of rats with rT3 produced changes in beta-adrenoreceptors similar to those seen in hypothyroid rats.     

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.     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Environmental salinity selectively modifies the outer-ring deiodinating activity of liver, kidney and gill in the rainbow trout

We here analyzed the effect of a mild hyperosmotic challenge on the activities of deiodinases type I (D1) and II (D2) in the trout liver, and D1 in kidney and gill, two organs involved in osmoregulation. FW-adapted immature rainbow trout were transferred to 5 per thousand SW and killed 0.5, 1, 2, 4, 8 12, 24 and 48 h post-transfer (PT). Fish maintained in FW served as controls. Hepatic, renal and branchial D1 and hepatic D2 activities were assessed as well as circulating levels of T(3), T(4) and cortisol. Hyperosmotic challenge elicited significant and sustained decreases in kidney D1 and liver D2 activities at 8 h PT, which returned to control values at 48 h PT. In contrast, liver and gill D1 activities exhibited no significant change throughout the study. Also, significant increases in circulating T(4) at 2-4 and 48 h PT were observed. Circulating T(3) remained unmodified until 24-48 h PT, when it rose sharply. Simultaneously, cortisol showed a trend towards increase during the initial 4 h PT, which attained significance at 48 h PT. The present findings demonstrate that a mild hypertonic challenge is sufficient to elicit responses in the trout thyroidal axis. Hormonal changes in the circulatory compartment are in accordance with those previously described for migratory salmonids. A novel aspect of our findings is the organ-specific differential response exhibited by ORD-enzymes when trout are exposed to a mildly different osmotic environment. Our findings further establish the uniqueness of fish thyroid physiology, and can be of value in further understanding the evolutionary aspects of this ORD family of deiodinases.     

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

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.     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.