Investigation of type I and type III iodothyronine deiodinases in rat tissues using N-bromoacetyl-iodothyronine affinity labels

Abstract

In the present study the hypothesis was tested that N-bromoacetyl-3,3′,5-[ 125I]triiodothyronine (BrAc[ 125I]T3) is a useful affinity label for both type I and type III iodothyronine deiodinases (ID-I and ID-III). Therefore, the microsomal fractions of various rat tissues were tested for ID-I and ID-III activities, and microsomal proteins were labeled with BrAc[ 125I]T3 and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I activities were found in liver, kidney and thyroid, and high ID-III activities in brain, in particular fetal brain, and placenta. SDS-PAGE of BrAc[ 125I]T3-labeled microsomes showed a prominent radioactive ∼27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a ∼32 kDa protein (p32) in brain, in particular fetal brain, and placenta. A good correlation was found between the affinity labeling of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents ID-III or a subunit thereof. After treatment of microsomes with 0.05% deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by BrAc[ 125I]T3, indicating that p32 is a transmembrane protein. Although 3,3′,5′-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily labeled with BrAc[ 125I]rT3. Labeling of p32 in rat brain microsomes by BrAc[ 125I]rT3 was not affected by addition of 100 μM unlabeled thyroxine (T4) or T3, whereas deiodination of [ 125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 μM T4 and T3, respectively. The relationship between p32 and ID-III was further questioned by findings of prominent BrAc[ 125I]T3 labeling of p32 in microsomes of spleen and fetal liver, which show very little ID-III activity. Peptide-mapping of p32 using trypsin or Staphylococcus aureus V8 protease yielded the same fragments in brain, placenta, spleen and fetal liver, suggesting that p32 is identical in all these tissues. In contrast to initial suggestions, therefore, we conclude that it is unlikely that the labeling of p32 by BrAc[ 125I]T3 represents the identification of ID-III.