Abstract
Extrathyroidal production of 3,3',5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5'-deiodinases. Type I 5'-deiodinase (5'D-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',5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5'D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5'D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5'D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5'D-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'-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'-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5'D-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'D-I showed coincident peaks of p27 and 5'-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'D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5'-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.
Highlights
Of the Ta found in the circulation and in thyroid hormone-responsive tissues and is most. abundant in rat liver and kidney
We have recently shown that BrAcT, or BrAcTB labels six to eight proteins in liver and kidney microsomal preparations with 75% of the affinity label associated with a 27-kDa protein [21, 22]
We show that BrAcTl is a potent, irreversible inhibitor of type I 5’-deiodinase and identify a 27-kDa integral membrane protein as a subunit of 5’D-I by BrAcT4 affinity labeling
Summary
Of the Ta found in the circulation and in thyroid hormone-responsive tissues and is most. abundant in rat liver and kidney. We used the bromoacetyl derivatives of T4 (iV-bromoacet.yl-[‘251]L-thyroxine, BrAcT4) and Tt (iV-bromoacetyl-[‘261]3,3’,5-triiodothyronine, BrAcTB) as alkylating affinity labels to identify 5’D-I-related protein(s). BrAcT4 and BrAcTs rapidly and irreversibly inactivated 5’D-I activity in liver and kidney microsomes. 5’D-I preparations showed that -80% of the affinity label was incorporated into a protein with a M, of. Hyper- and hypothyroidism-induced increases and decreases in 5’D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled ~27. 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5’D-I content. 114, and the 16th Annual Meeting of the European Thyroid Association ((1987) Ann. Endocrinol. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “aduertisement”
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