Iodothyronine 5′-Deiodinase from Rat Kidney: Substrate Specificity and the 5′-Deiodination of Reverse Triiodothyr onine *

Abstract

Renal membranes (crude microsomal fraction) which catalyze the thiol-dependent outer ring deiodination of LT₄ with the formation of L-T₃ are also capable of the 5′-deiodination of rT₃ with the production of equivalent amounts of I and 3,3′-T₂. rT₃ deiodination was followed by measuring the amount of ¹²⁵I released from ¹²⁵I-labeled (3′ or 5′-) L-rT₃; iodide and iodothyronines were separated by a rapid method using Dowex 50W cation exchange columns. Iodide release was proportional to tissue concentration, was heat labile, and was unaffected by methylmercaptoimidazole. The reaction had a rapid phase (∼ 10–15 sec) which was unaffected by thiols and a slower thiol-dependent phase which was linear with time. Double reciprocal plots of product formation vs. rT₃ concentration at fixed thiol concentrations yielded a family of parallel lines. Parallel lines were also obtained when the thiol concentration was variable and the rT₃ concentration was fixed. These patterns of initial velocity kinetics resemble those obtained with L-T₄ as a substrate. Limiting Michaelis constants K_a for rT₃ and K_b, for dithiothreitol were 0.46 μM and 8.1 mM, respectively, and the V₁ was 0.99 nmol I released protein-min; the K_b and V₁ were at least 15 times greater than comparable values for T₄ 5′ deiodination whereas the K_a values for the iodothyronine substrates were nearly equivalent. Pretreatment of the renal microsomal fraction with 10 μ 6-propy1-2-thiouracil and increasing concentrations of rT₃ resulted in increasing degrees of persistent 6-propyl-2-thiouracil inhibition of both T₄ and rT₃ 5′-deiodination reactions. The substrates, T₄ and rT₃, were found to protect iodothyronine 5′-deiodinase from inactivation by iodoacetate. These findings are consistent with the idea that the same enzymecatalyzes the 5′-deiodination of both T₄ and rT₃, that measuring I release from rT₃ is an effective method for assay of the enzymatic activity, and that thiol availability may determine the relative rates of T₄ and rT₃ metabolism via this enzyme.