Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell hemogenate from monkey

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3,5-¹²⁵I]triido-l-thyronine or 3-[3′5′-¹²⁵I]triido-l-thyronine (phenolic ring-labeled ‘reverse’ triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, 0.9×～1 cm) to measure ¹²⁵I^?. Both deiodinases were destroyed by boiling for 1 min.Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1–5 mM), and slightly by 5 mM β-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 mM) and inhibited by Mg²⁺ (5 mM). Methylmercaptoimidazol and Mg²⁺, Ca²⁺ and Mn²⁺ (0.1–1.0 mM) had little or no effect on either reaction, but Zn²⁺ (0.1 mM) strongly inhibited both.Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10μM, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3′,5′-triiodo-l-thyronine-(rT₃) > l-thyroxine(T₄) > 3,4,3′-triido-l-thyronine(T₃), whereas for the nonphenolic ring deiodinase the order is T₃ > T₄ > rT₃. Diiodotyrosine did not affect their deiodination.