Sequential deiodination of thyroxine in rat liver homogenate

Visser, Theo J.; Fekkes, Durk; Docter, R.; Hennemann, G

doi:10.1042/bj1740221

Cited by 94 publications

(42 citation statements)

References 27 publications

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“…As is well known from both in vivo and in vitro data (32,33), PTU efficiently inhibits outer ring deiodination of T4 and T3. PTU also has a slight inhibitory ef-fect on inner ring deiodination of T4 and T3 (34,35), which results in decreased serum T3 concentration with reciprocally increased rT3 concentration (36). These inhibitory effects on T4 metabolism increase T4 metabolism through the conjugating pathways (37).…”

Section: Resultsmentioning

confidence: 99%

“…[3,5-125I]T4 (-2 mCi/,Lg) and [3,]T3 were synthesized according to Cahnmann's methods (19,20). All radioactive tracers stored >10 d (in ethanol at 4°C) were purified by ion-exchange column chromatography (0.9 x 10 cm, Dowex 50W-X4, [30][31][32][33][34][35] ,um, Bio-Rad Laboratories, Richmond, Calif.), followed by passing through a short column (0.9 x 1.0 cm) of Dowex 50W-X4 to remove 125I-as described previously (17). Other materials were 6-n-propyl-2-thiouracil (PTU) (Sigma Chemical Co., St. Louis, Mo.…”

Section: Methodsmentioning

confidence: 99%

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Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

Sato¹,

Robbins²

1981

J. Clin. Invest.

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A B S T R A C T Primary cultured adult rat hepatocytes were used to study the regulation of thyroid hormone deiodination. Control studies showed that these cells survived for at least 4 d, during which time they actively deiodinated both the phenolic (5'-) and nonphenolic (5-) rings of L-thyroxine (T4),3,5,3'-triiodo-L-thyronine, and 3,3',5'-triiodothyronine. Increasing the substrate concentration caused a decrease in fractional iodide release and a corresponding increase in conjugation with sulfate and glucuronide. Propylthiouracil strongly inhibited the 5'-deiodinase activity and caused only a slight decrease in 5-deiodinase activity. Thus, these monolayer-cultured cells preserved many of the properties of normal hepatocytes. Incubation with combinations of insulin, glucagon, and/or glucose for 5 h showed that insulin stimulated T4 5'-deiodination, whereas glucagon inhibited the insulin stimulation but had no effect in the absence of insulin. Glucose had no effect and did not alter the effect of the hormones. The insulin-enhanced deiodination increased between 1 and 5 h, which suggests that the previous inability to demonstrate an insulin effect was due to the short survival of the in vitro liver systems used in those studies. The present data suggest that the inhibition of T4 5'-deiodination observed during fasting, and its restoration by refeeding, mnay be related to the effects offeeding on insulin and glucagon release rather than on glucose per se.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

Sato¹,

Robbins²

1981

J. Clin. Invest.

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“…1, A The ping-pong reaction mechanism is usually diagnosed by finding parallel double reciprocal plots of initial rate data and has been experimentally verified for the kidney and liver enzyme in a number of laboratories (10, 11), including our own (3). It must be emphasized, however, as Cleland has done (25,26), that the parallel initial velocity patterns in ping-pong mechanisms are observed only when the modified enzyme form generated after the release of the first product ( (27) and by Visser et al (28)], then in presence of excess PTU, the reaction will be diverted towards the formation of the PTU-complex (step 4) and the apparent first-order rate constant for the reaction of enzyme and substrate to give iodide will be nearly zero (ignoring the minute amount of iodide generated during the first catalytic cycle). Raising DTT levels will divert the reaction flux from the dead-end formation of the PTUcomplex (step 4) (or the spontaneous hydrolysis of the E.. * I complex) to thiol-dependent production of iodide (step 3) and thus increase the apparent rate of iodide generation (without affecting the true rate of combination between E and S).…”

Section: Discussionmentioning

confidence: 99%

Iodothyronine 5'-deiodinase in rat kidney microsomes. Kinetic behavior at low substrate concentrations.

Goswami¹,

Rosenberg²

1984

J. Clin. Invest.

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Abstract. The thiol-activated enzymatic outerring monodeiodination of iodothyronines by rat kidney microsomes at low (nanomolar) substrate concentrations shows an apparently sequential reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol, a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 MAM) and iopanoic acid (K1 = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile that is concave downward with a Td of -20'C. In contrast, the activity at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile. Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine (rT3) concentrations (0.1 nM to 10 uM) suggested the presence of two enzymatic activities, with apparent Michaelis constants (Kin) of 0.5 ,M and 2.5 nM. Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100 ,M propylthiouracil also revealed an operationally distinct enzymatic activity with Km's of 2.5 and 0.63 nM and maximum velocities (Vmax'S) of 16 and 0.58 pmol/mg protein per h for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well characterized high Km enzyme and suggest that at circulating levels of free T4 the postulated low K. enzyme could be physiologically important.

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“…Nonradioactive iodothyronines were obtained from Henning Berlin R&D (Berlin, Germany) and [3 ,5 -125 I]rT3 was prepared by radioiodination of 3,3 -diiodothyronine (T2) as described (Visser et al 1978 125 I]T3 as described (Mol et al 1984). HPLC analysis demonstrated that the purity of BrAc[…”

Section: Methodsmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

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Sequential deiodination of thyroxine in rat liver homogenate

Cited by 94 publications

References 27 publications

Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

Iodothyronine 5'-deiodinase in rat kidney microsomes. Kinetic behavior at low substrate concentrations.

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

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