Affinity labeling of rat liver and kidney type I 5\u27-deiodinase. Identification of the 27-kDa substrate binding subunit

Abstract

Extrathyroidal production of 3,3\u27,5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5\u27-deiodinases. Type I 5\u27-deiodinase (5\u27D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3\u27,5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5\u27D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5\u27D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5\u27D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5\u27D-I substrates and inhibitors specifically blocked affinity labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3\u27-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5\u27-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5\u27D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled p27. BrAcT3 was a less effective affinity label for p27 and minor labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5\u27D-I showed coincident peaks of p27 and 5\u27-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5\u27D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5\u27-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3