Reaction of the type III iodothyronine deiodinase with the affinity label <i>N</i>‐bromoacetyl‐triiodothyronine

Schoenmakers, Christian H.H.; Pigmans, Ingrid G.A.J.; Kaptein, Ellen; Darras, Veerle; Visser, Theo J.

doi:10.1016/0014-5793(93)80449-5

Cited by 11 publications

(5 citation statements)

References 18 publications

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“…Affinity labeling with BrAc[ 125 I]T 3 was also used for the quantitation of D1 enzyme expression levels by saturation analysis, allowing estimation of the D1 substrate turnover number in microsomal preparations and homogenates of transfected cells (41,50). With regard to affinity labeling of D3 with BrAcT3 results have been confusing (51)(52)67). Initial experiments with brain and placenta microsomes revealed a good correlation between affinity labeling of a 32-kDa protein (p32) and inactivation of D3 by unlabeled BrAcT3 (51,52).…”

Section: Discussionmentioning

confidence: 99%

“…With regard to affinity labeling of D3 with BrAcT3 results have been confusing (51)(52)67). Initial experiments with brain and placenta microsomes revealed a good correlation between affinity labeling of a 32-kDa protein (p32) and inactivation of D3 by unlabeled BrAcT3 (51,52). However, when p32 was also detected in tissues with low or unde-tectable D3 activity and p32 labeling was not prevented by substrates of D3, it was concluded that p32 does not represent D3 protein (51,52).…”

Section: Discussionmentioning

confidence: 99%

“…Initial experiments with brain and placenta microsomes revealed a good correlation between affinity labeling of a 32-kDa protein (p32) and inactivation of D3 by unlabeled BrAcT3 (51,52). However, when p32 was also detected in tissues with low or unde-tectable D3 activity and p32 labeling was not prevented by substrates of D3, it was concluded that p32 does not represent D3 protein (51,52). An alternative explanation for the inconsistencies might be that in one-dimensional SDS-PAGE, the low abundance D3 protein comigrates with an abundant p32 protein not related to D3 (52,67).…”

Section: Discussionmentioning

confidence: 99%

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Substitution of Cysteine for Selenocysteine in the Catalytic Center of Type III Iodothyronine Deiodinase Reduces Catalytic Efficiency and Alters Substrate Preference

2003

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Human type III iodothyronine deiodinase (D3) catalyzes the conversion of T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring deiodination. Like types I and II iodothyronine deiodinases, D3 protein contains selenocysteine (SeC) in the highly conserved core catalytic center at amino acid position 144. To evaluate the contribution of SeC144 to the catalytic properties of D3 enzyme, we generated mutants in which cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were transfected with expression vectors encoding D3wt, D3Cys, or D3Ala protein. Kinetic analysis was performed on homogenates with dithiothreitol as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4) increased 100-fold. The D3Ala protein was enzymatically inactive. Semiquantitative immunoblotting of homogenates with a D3 antiserum revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are expressed relative to D3wt protein. The relative substrate turnover number of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4) deiodination, compared with D3wt enzyme. Studies in intact COS cells expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under in situ conditions. In conclusion, the SeC residue in the catalytic center of D3 is essential for efficient inner-ring deiodination of T(3) and in particular T(4) at physiological substrate concentrations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Substitution of Cysteine for Selenocysteine in the Catalytic Center of Type III Iodothyronine Deiodinase Reduces Catalytic Efficiency and Alters Substrate Preference

2003

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“…D3 is found in many tissues, predominantly in the brain and in the placenta, and levels usually remain stable over a wide range of metabolic states Chanoine et al, 1993;Schoenmakers et al, 1993Schoenmakers et al, , 1995. As shown in Table 2, there was no effect of MeI on brain D3 activity in the exposed rats.…”

Section: D3 Analysismentioning

confidence: 84%

“…Results (mean ± SD) are presented as percent of control, which is the D2 activity in the brain homogenates from rabbits not exposed to MeI. quantities of D3 that likely protect the fetal compartment from toxic levels of maternal iodothyronines Chanoine et al, 1993;Schoenmakers et al, 1993Schoenmakers et al, , 1995. Indeed, the rabbit placenta contains ~10-fold higher D3 levels than were observed in the rat brain (195.2 ± 63.1 fmol T2 produced/mg protein/h).…”

Section: D3 Analysismentioning

confidence: 94%

Effect of methyl iodide on deiodinase activity

Farwell¹,

Leonard

2009

Inhalation Toxicology

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Methyl iodide (MeI) has been proposed as an alternative for methyl bromide in pre-plant soil fumigation applications that does not affect stratospheric ozone. Preliminary studies in rabbits noted fetal resorptions if the pregnant does were exposed to MeI during a critical period during gestation. In addition, abnormalities in thyroid hormone parameters were also observed in animals exposed to MeI. Since monodeiodination is the major metabolic pathway of the thyroid hormones, we examined the effect of MeI on deiodinase activity as a possible etiology for the alteration in thyroid hormone parameters and ultimate fetal demise. In vitro studies using tissue microsomes and cell culture showed that MeI has no effect on type I 5'-deiodinase (D1) or type II 5'-deiodinase (D2) at physiologically relevant concentrations. At high concentrations (>10 mM,>10,000 ppm), MeI caused a nonspecific inactivation of D1 and D2. Analysis of D1 and D2 activity in rats exposed by inhalation to increasing concentrations of MeI showed a significant decrease in enzyme activity at 100 ppm, while brain type III 5'-deiodinase (D3) was unaffected by MeI at the exposures studied. While the drop in D1 can be explained by the induction of a hypothyroid state in the exposed rats, there is no clear explanation for the fall in D2 levels. In the rabbit studies, there was a significant decrease in kidney D1 in the adult rabbits exposed to 20 ppm MeI. However, there was no effect on liver D1, brain D2, or placental D3 in the MeI-exposed rabbits. Similarly, there was no effect of MeI on fetal D1 or D2 activity. The lack of a significant direct effect of MeI on deiodinase activity and the absence of a change in placental or fetal deiodinase activity make it unlikely that alterations in deiodinase activity plays a role in the fetal resorptions in the MeI-exposed rabbits.

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Thyroxine 5-deiodinase

2009

Class 1 · Oxidoreductases

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Reaction of the type III iodothyronine deiodinase with the affinity label N‐bromoacetyl‐triiodothyronine

Cited by 11 publications

References 18 publications

Substitution of Cysteine for Selenocysteine in the Catalytic Center of Type III Iodothyronine Deiodinase Reduces Catalytic Efficiency and Alters Substrate Preference

Substitution of Cysteine for Selenocysteine in the Catalytic Center of Type III Iodothyronine Deiodinase Reduces Catalytic Efficiency and Alters Substrate Preference

Effect of methyl iodide on deiodinase activity

Thyroxine 5-deiodinase

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