Characteristics of Iodothyronine Tyrosyl Ring Deiodination by Rat Cerebral Cortical Microsomes*

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Abstract
We studied the biochemical properties of tyrosyl ring (5-) deiodination of L-T3 and L-T4 by cerebrocortical microsomes from euthyroid rats. Incubations contained radioiodinated L-T3) or L-T4 and dithiothreitol (DTT), and products were analyzed by paper chromatography. The pH maximum was 7.5 for 5-deiodination of both T3 and T4. Increasing the DTT concentration from 5 to 200 mM caused a progressive increase in the 5- deiodination rate of 2 nM T3, but at 500 mM DTT the rate decreased. When three DTT concentrations (5.25, 10.1, and 20.1 mM) were used, double reciprocal plots of 5-deiodination rate as a function of T3 concentration (from 0.3–40 nM) showed a sequential type kinetic pattern, with a limiting Kn of 1.7 nM L-T3. Cerebral microsomes from 4-day-old rats had an apparent Km for L-T3 similar to that of cerebrocortical microsomes from adult rats, but the neonatal tissues had a 3.5- to 10-fold higher apparent maximum velocity. At 50 mM DTT, L-T3 and L-T4 each inhibited 5-deiodination of the other. The apparent Km and Ki for L-T3 were 5.5 and 8.0 nM, respectively, the apparent Km and Ki for L-T4 were 37 and 48 nM, respectively, and the apparent maximum velocities for 5-deiodination of T3 and T4, respectively, were 134 and 144 fmol/min/mg protein. There was dose-dependent inhibition of L-T3 5-deiodination by tetraiodothyroacetic acid, D-T3, tetraiodothyroacetic acid, 3,5-L-diiodothyronine, 3,3′-L-diiodothyronine, iopanoic acid, rose bengal, L-rT3, erythrosine, bromphenol blue, and anilinonaphthalenesulfonic acid (in decreasing order of potency). There was no effect on reaction rate by addition of 3′-L-monoiodothyronine, L-thyronine, DIT, amiodarone, dicoumarol, Nal, propylthiouracil (PTU), sodium salicylate, sodium fluoride, EDTA, CaCl2, or MgCl2. These data demonstrate that the properties of the rat brain iodothyronine tyrosyl ring deiodinase differ from those of both the PTU-sensitive and PTU-insensitive pathways of 5′-deiodination in rat brain and also differ from the properties of the iodothyronine 5-deiodinase in rat liver.