Properties of a Rat Tissue Iodothyronine Deiodinase and Its Natural Inhibitor^*

Abstract

B-ring deiodination of L-thyroxine (T4) and L-triiodothyronine (Ta) by rat liver is enzymatic in nature, but the deiodinase activity in crude tissue homogenates is largely blocked by a naturally occurring inhibitor. The inhibitor is responsible for the time lag observed before deiodination takes place, and it is also responsible for the relative heat stability, pH dependence, and substrate inhibition of the system. All of these effects disappeared after the inhibitor was removed by dialysis. The specific activity of deiodinase was highest in liver, kidney, and heart microsomes, and the concentration of deiodinase remained the same in tissues of hypo-, eu-, and hyperthyroid animals. However, tissue levels of the inhibitor were highest in hypothyroid and lowest in hyperthyroid animals, suggesting that the natural inhibitor may play some role in metabolic control. The natural T he earliest report of an in citro system for the enzymatic deiodination of L-thyroxine (T4) and Ltriiodothyronine (T3) was published in 1955 by Sprott and Maclagan. Since then, various aspects of the reaction have been studied, but the published results and conclusions are confusing. An inhibitor that prevents the deiodination reaction from following established enzyme kinetic theory is probably the major cause of this confusion (Stanbury et al., 1960;Wynn and Gibbs, 1962;Nakagawa and Ruegamer, 1964;Yamamoto, 1964). However, a wide variation in substrate concentrations, pH optima, enzyme sources, cofactors, and assay techniques were used, and both enzymatic and nonenzymatic deiodination were observed. It has been suggested that some of the deiodination systems described may be artifactual (Job-Buzo et al., 1966). In view of the current lack of clarity as to the mechanism and enzymatic nature of the iodothyronine deiodination reaction, a detailed and systematic study of the reaction was made, and the results are summarized below.inhibitor was found in the soluble portion of the cell and was readily dialyzable. The identity of the inhibitor remains unknown, but a number of reducing substances such as ascorbate, reduced nicotinamideadenine dinucleotide, reduced nicotinamide-adenine dinucleotide phosphate, cysteine, and reduced glutathione markedly inhibited deiodination. Therefore, the inhibitor may be a reducing substance that keeps either the active site on the enzyme or the substrate in the reduced form so that deiodination cannot proceed. Such a concept presupposes that deiodination is intimately concerned with oxidation of the thyronine ring structure, probably through the formation of a quinone. Removal of B-ring iodines may actually be secondary to oxidation of the phenol group, and the natural inhibitor may interfere with this oxidative step. Experimental ProcedureAnimals and Tissue Homngenates. Holtzman strain male rats weighing 100-110 g were maintained on a Purina chow diet for at least 3 weeks prior to the experiment. The animals were sacrificed by decapitation, and 2.00-g samples of tissue were quickly removed and homogenized in a s...

Tissue iodoprotein formation during the peripheral metabolism of the thyroid hormones

“…The enzymatic nature of the deiodination of T4 and T3 by hepatic microsomes (12,27,28) and the increased rate of deiodination by microsomes from PB-treated rats have been demonstrated previously (20). Fig.…”

“…Furthermore, the increased rate of deiodination by PB-microsomes was accompanied by a proportional increase in the rate of microsomal NEI formation. Other workers have also reported the association of chromatographic origin radioactivity (presumably NEI) and iodide formation during the metabolism of the iodothyronines by liver homogenates (9,10,12) and microsomes (11). The lack of NEI formation during the metabolism of 3-monoiodotyrosine (MIT) suggests the specificity of this pathway for the iodothyronines.…”

“…Although iodoprotein formation during thyroid hormone metabolism by various tissues in vitro has been frequently reported (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), the results of some of these studies must be queried since the incubation systems which were employed were supplemented with flavin mononucleotide (FMN). This cofactor in the presence of light has been shown to produce deiodination of iodothyronines and radioactivity remaining at the origin of chromatograms in the absence of any tissue components (13,14).…”

“…Moreover, the addition of a variety of proteins which have no deiodinating capability alone greatly accelerates this FMN effect (15). In several reports, however, in vitro iodoprotein formation was clearly demonstrated in incubation systems which did not require FMN (8,(9)(10)(11)(12). Fewer data are available on in vivo iodoprotein formation during thyroid hormone metabolism.…”

Iodothyronine Metabolism in Rat Liver Homogenates

“…Rates of removal of the 5'-and 5-iodine of T4 are approximately equal in humans and rats (3,4), but these processes do not occur randomly. Decreased serum T3 concentrations, resulting from decreased peripheral 5'-deiodina-Many tissues, including liver, kidney, heart, skeletal muscle, leukocytes, and fibroblasts, are capable of deiodinating T4 in vitro (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). The techniques used to demonstrate T4 deiodination in these studies included incubation of tissue homogenates, tissue slices, dispersed cells, and whole organ perfusion.…”