Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5′-deiodinase

Author:

Bhat G B1,Iwase K1,Hummel B C W1,Walfish P G1

Affiliation:

1. The Thyroid Research Laboratory, Mount Sinai Hospital Research Institute, and Department of Medicine, Endocrine Division, Mount Sinai Hospital, University of Toronto Medical School, Toronto, Ont. M5G lX5, Canada

Abstract

The properties and kinetic characteristics of a non-GSH NADPH-dependent cofactor system activating rat hepatic and renal 5′-deiodinase (5′-DI), which we have previously demonstrated with partially purified cytosol Fractions A and B [Sawada, Hummel & Walfish (1986) Biochem. J. 234, 391-398], were examined further. Although microsomal fractions prepared from either rat liver or kidneys could be activated by crude cytosol Fractions A and B from those tissues as well as from rat brain and heart, a homologous hepatic or renal system was the most potent in producing 5′-deiodination of reverse tri-iodothyronine (rT3). At nanomolar concentrations both rT3 and thyroxine (T4) were deiodinated but with a much greater substrate preference for rT3 than for T4. However, at micromolar concentrations of these substrates no activation of 5′-DI could be detected. In this deiodinative system, T4 and tri-iodothyronine (T3) competitively inhibited 5′-deiodination of rT3. Dicoumarol, iopanoate, arsenite and diamide were also inhibitory to the activation of hepatic or renal 5′-deiodination by this cofactor system. Purification of cofactor components in hepatic crude cytosolic Fractions A and B to near homogeneity, as assessed by their enzymic and physical properties, indicated that these co-purified with and were therefore identical with thioredoxin reductase and thioredoxin respectively, and accounted almost entirely for the observed activation of rT3 5′-DI. When highly purified liver cytosolic thioredoxin reductase and thioredoxin were utilized to determine the kinetic characteristics of the reaction, evidence for a sequential mechanism operative at nanomolar but not micromolar concentrations of rT3 and T4 was obtained. The Km for rT3 was 1.4 nM. Inhibition by 6-n-propyl-2-thiouracil (Ki 6.7 microM) was competitive with respect to thioredoxin and non-competitive with respect to rT3, whereas inhibition by T4 (Ki 1.3 microM) was competitive. Since rT3 is a potent inhibitor of T4 5′-deiodination, this thioredoxin system activating deiodination of rT3 may play an important role in regulating the rate of intracellular production of T3 from T4.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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