title: Affinity labeling of rat liver and kidney type I 5'-deiodinase. Identification of the 27-kDa substrate binding subunit.
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.
authors.affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
authors.forename: J
authors.initials: J
authors.lastname: Köhrle
authors.forename: U B
authors.initials: UB
authors.lastname: Rasmussen
authors.forename: D M
authors.initials: DM
authors.lastname: Ekenbarger
authors.forename: S
authors.initials: S
authors.lastname: Alex
authors.forename: H
authors.initials: H
authors.lastname: Rokos
authors.forename: R D
authors.initials: RD
authors.lastname: Hesch
authors.forename: J L
authors.initials: JL
authors.lastname: Leonard
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002850:Chromatography, Gel; D006980:Hyperthyroidism / Q000201:enzymology; D007037:Hypothyroidism / Q000201:enzymology; D007453:Iodide Peroxidase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011441:Propylthiouracil / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D011919:Rats, Inbred Strains; D012016:Reference Values; D013974:Thyroxine / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D000345:Affinity Labels; D007527:Isoenzymes; D046911:Macromolecular Substances; D011441:Propylthiouracil; D007453:Iodide Peroxidase; D013974:Thyroxine