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

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 Ka for rT3 and Kb, for dithiothreitol were 0.46 μM and 8.1 mM, respectively, and the V1 was 0.99 nmol I released protein-min; the Kb and V1 were at least 15 times greater than comparable values for T4 5′ deiodination whereas the Ka 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.