Characteristics of Outer Ring (5′- or 3′-) Monodeiodination of 3′,5′- and 3,3′-Diiodothyronine: Evidence Suggesting One Outer Ring Monodeiodinase for Various Iodothyronines*

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Abstract
Previous studies revealing marked similarities in the activities of outer ring monodeiodination of T4 and rT3 by liver or kidney have suggested that these processes are mediated by the same enzyme. It is not known, however, whether the same, similar, or different enzyme (s) is involved in the removal of iodine from the outer ring of other iodothyronines. This study examines the processes of extrathyroidal monodeiodination of 3′5′-diiodothyronine (3′,5′-T2) and 3,3′-T2 in the outer ring to 3′- monoiodothyronine (3′-T1) and 3-T1, respectively, and describes their responses to several manipulations. 3′5′-T2 (∼8 × 108 M) or 3,3′-T2 (∼3 × 107 M) was incubated in 0.1 M Tris buffer (pH 7.4) with various rat tissue homogenates (∼0.13 geq) for 3 min (in the case of 3′,5′-T2) or 30 min (in the case of 3,3′-T2) at 37 C, and the amount of T1 generated (3′-T1 from 3′,5′-T2 and 3-T1 from 3,3′-T2) during incubation was quantified by pecific RIA of ethanol extracts of incubation mixtures. The radioactive ligand in the 3′-T1 RIA was 125I-labeled 3′-T1; in the 3-T1 RIA it was 3-T1-[125I]iodohistamine prepared by conjugation of 3-T1 to [125I]iodohistamine. The liver and kidney produced more T1 from outer ring deiodination of both T2s than various other tissues. Studies using liver homogenate indicated that onodeiodination of both T2s was influenced in a similar manner by the concentration of the homogenate, duration of incubation, substrate (T2) concentration, pH of incubation, and temperature of incubation. The T2-monodeiodinating activities were unaffected by large concentrations (≥1 mM) of potassium iodide, methimazole, and ascorbic acid. However, they were both stimulated by dithiothreitol and inhibited in a dose-dependent manner by (in order of relative potency on a molar basis, maximal to minimal) rT3, ipodate, T4, propylthiouracil, diamide, 8-anilino-l-naphthalene sulfonic acid, and salicylate. The apparent Km values for monodeiodination of 3′5′-T2 (0.5 μM) and 3,3′- T2 (0.45 μM) were also similar, but the Vmax for monodeiodination of 3′5′-T2 (390 pg/mg protein-min) was substantially higher than that of 3,3′-T2 (33 pg/mg protein min). Among the usual subcellular fractions of the liver homogenate, microsomes were more potent and cytosol was less potent in deiodinating both T2s than mitochondria or the starting homogenate. However, the plasma membrane fraction of the homogenate, prepared separately by sucrose density gradient, was the single most active subcellular fraction for both monodeiodinations. Starvation of the rat for 2 or 4 days was associated with a significant reduction in T2-monodeiodinating activity in the liver homogenate. The various data suggest that 1) 3′5′-T2 (to 3′-T1) and 3,3′-T2 (to 3- T1) monodeiodinating activities are enzymatic in nature; 2) these processes are very similar to each other and to T4 and rT3 outer ring monodeiodinating activities in many characteristics, including organ distribution, subcellular location, and their responses to various manipulations; and 3) there may just be one outerring monodeiodinase acting on various iodothyronines.