Ligand-induced inactivation of type I iodothyronine 5'-deiodinase: protection by propylthiouracil in vivo and reversibility in vitro.
The inactivation of the phenolic ring deiodinases by natural substrates and iodinated contrast agents such as iopanoic acid (IOP) is a principal mechanism regulating the peripheral metabolism of thyroid hormones. The present studies were designed to investigate the cellular and biochemical mechanisms mediating this effect on type I 5'-deiodinase (5'DI) in rat liver and kidney. We reasoned that if inactivation requires the direct interaction of ligand (e.g. IOP) with 5'DI, then this effect should be blocked by a prior injection of 6n-propyl-2-thiouracil (PTU), which binds to an essential sulfhydryl group at the enzyme's active site and inhibits catalytic cycling. Experiments were performed using liver and kidney microsomes prepared from male rats injected 2.5 or 6 h earlier with various combinations of PTU, IOP, and/or vehicle. In initial studies, conditions were defined whereby active 5'DI could be regenerated in vitro from PTU-injected animals. This involved the treatment of microsomes with 50 mM dithiothreitol, pH 7.0, before assay. After this reductive treatment, 5'DI activity in both liver and kidney microsomes from rats previously injected with PTU, or the combination of PTU followed 30 min later by IOP, was equivalent to that in vehicle-injected control animals, whereas activity was decreased 60% in microsomes from animals injected with only IOP. This demonstrates that PTU protects 5'DI against ligand-induced inactivation, suggesting that this process is initiated by substrate binding to the enzyme's active site. In other experiments, liver microsomes from IOP-injected animals were pretreated with 100 mM dithiothreitol, pH 9.0, then washed, and 5'DI activity was determined. Under these stringent reducing conditions, 5'DI activity was completely recovered in microsomes from rats injected with IOP 2.5 h earlier, but only partially recovered 6 h after the IOP injection. Thus, ligand-induced inactivation of 5'DI may involve initially a reversible oxidation of the enzyme, followed by its irreversible inactivation.
St Germain
D L
DL
Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03756.
Croteau
W
W
D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009928:Organ Specificity; D011441:Propylthiouracil / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values; D013974:Thyroxine / Q000494:pharmacology
D008024:Ligands; D011441:Propylthiouracil; D007453:Iodide Peroxidase; D013974:Thyroxine; D004229:Dithiothreitol