Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver

Brinke, D Auf Dem; Hesch, R. D.; Köhrle, Josef

doi:10.1042/bj1800273

Cited by 34 publications

(7 citation statements)

References 18 publications

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“…There is general agreement that D1 is an integral membrane protein but different cellular localizations have been found. In rat kidney, D1 is present in the plasma membrane (Leonard & Rosenberg 1978, Leonard et al 1991, while in rat liver D1 may be present in the ER with its active site oriented to the cytoplasm (Auf dem Brinke et al 1979, Fekkes et al 1979, Toyoda et al 1995b. By immunofluorescence confocal microscopy the D1 protein was localized to the plasma membrane of transiently transfected COS cells (Baqui et al 2000).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…(1) Both 5-and 5'-deiodinase activities have been localized in the endoplasmic reticulum (Fekkes et al, 1979;Auf dem Brinke et al, 1979, 1980.…”

Section: Discussionmentioning

confidence: 99%

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

Fekkes¹,

Hennemann²,

Visser³

1982

Biochemical Journal

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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.

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“…The mixture was shaken for 10 min at 4°C and then centrifuged as published earlier (Hesch et al 1975). The supernatant was taken for radioimmunological determination of T3, rift and 3,3'-Tî as previously described (Auf dem Brinke et al 1979). Iodothyronines concentration determined after 20 min incubation were corrected for the iodothyronines present at time zero.…”

Section: Methodsmentioning

confidence: 99%

Autoregulation of 3, 3′,5′-triiodothyronine production by rat liver microsomes

Kamiński

Köhrle

Ködding

et al. 1981

Acta Endocrinologica

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Conversion of thyroxine (T4) to 3,3\m=' \,5\m=' \-triiodothyronine (rT3) was studied in rat liver microsomes. Addition of rT3 at a physiological concentration to the incubation medium inhibited the deiodination of thyroxine to rT3. With a concentration of rT3 greater than 37.6 nM no net rT3 production at pH 8.0 was observed. Further increases in rT3 concentration resulted only in degradation of added rT3 and no net synthesis of rT3 from T4 could be detected. The inhibitory effect of rT3 upon its own production from T4 was pH dependent, 5 fold lower amounts of hormone being required to inhibit completely rT3 production at pH 7.4 than at pH 8.0. With the same experimental conditions no significant effect of rT3 on the conversion of T4 to 3,5,3\m='\-triiodothyronine (T3) could be observed at pH 8.0 with all concentrations of added iodothyronine. A linear production of 3,3\m=' \-T2 from added rT3 was determined over the whole range of rT3 concentration, suggesting a lack of saturation of deiodinating enzyme. Binding of rT3 by anti-rT3 antibody added to the incubation mixture enhanced rT3 production from T4 by protecting rT3 from being degraded and/or diminishing the inhibitory effect of this iodothyronine on its own production. It was concluded that rT3 influenced its own production and that this effect may represent an important autoregulatory process in the iodothyronine metabolism.

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Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver

Cited by 34 publications

References 18 publications

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

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

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

Autoregulation of 3, 3′,5′-triiodothyronine production by rat liver microsomes

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