Investigation of type I and type III iodothyronine deiodinases in rat tissues using N-bromoacetyl-iodothyronine affinity labels.
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 approximately 27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a approximately 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 microM unlabeled thyroxine (T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Schoenmakers
C H
CH
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Pigmans
I G
IG
Visser
T J
TJ
D000345:Affinity Labels; D000818:Animals; D005260:Female; D066298:In Vitro Techniques; D007453:Iodide Peroxidase / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D010449:Peptide Mapping; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D014284:Triiodothyronine / Q000031:analogs & derivatives*
D000345:Affinity Labels; D014284:Triiodothyronine; C036219:N-bromoacetyl-3,3',5-triiodothyronine; D007453:Iodide Peroxidase