A STUDY OF THE CHARACTERISTICS OF THE RAT PLACENTAL IODOTHYRONINE 5-MONODEIODINASE - EVIDENCE THAT IT IS DISTINCT FROM THE RAT HEPATIC IODOTHYRONINE 5'-MONODEIODINASE

Recent studies have demonstrated that rat liver type I iodothyronine 5'-monodeiodinase (5'-MD) characteristically contains selenocysteine. The present study was undertaken to characterize rat placental type III iodothyronine 5-MD and to compare it with 5'-MD. Solubilized rat placental microsomes were delipidated by carboxymethyl cellulose-Sephadex chromatography. Phospholipids and proteins were recovered in two distinct peaks, which did not show 5-MD activity. 5-MD activity was recovered fully, however, by combining the two components (phospholipids and protein) and partially after the addition of exogenous phospholipids to protein. Tissue selenoproteins were labeled by injection of radioactive selenium (Se-75; 50-mu-Ci, iv; on days 5, 10, and 15 of gestation) to pregnant rats. Subcellular fractions of maternal and fetal tissues were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. No specific seleno-labeled proteins were evident in the microsomes of placenta or maternal or fetal brain. A 27- to 29-kilodalton (kDa) band previously suggested to be 5'-MD was observed, however, in maternal liver and kidney microsomes. Aurothioglucose inhibited rat placental 5-MD, but the dose required for 50% inhibition was over 50-fold greater than that for Se-containing hepatic 5'-MD (430 vs. 8 nM). The mechanism of the inhibition was noncompetitive for 5-MD, whereas it was competitive for 5'-MD. A synthetic peptide of 16 amino acids corresponding to the carboxy-terminal portion of 5'-MD was synthesized, and rabbits were immunized with the peptide-BSA conjugate. Western blot studies using the rabbit antiserum showed one specific 29-kDa band in rat liver microsomes. However, no specific bands were observed in 5-MD-rich placental or fetal brain microsomes. Bromoacetyl T3 (BrAcT3) was a potent inhibitor of rat placental 5-MD. Affinity labeling of solubilized rat placental microsomes with [I-125]BrAcT3 showed a predominant band of 31 kDa, distinct from the 27- to 29-kDa band found in liver and kidney. The labeling of the 31-kDa band was enhanced by 10 mM dithiothreitol, inhibited 60% by 150-mu-M T3, and prevented by 40-mu-M aurothioglucose. A dominant affinity-labeled 31-kDa band was also observed in fetal brain microsomes. Some tissues without 5-MD activity (testes and spleen) also showed weak binding. We conclude that rat placental 5-MD 1) requires phospholipids for enzymatic activity, 2) is not a seleno-containing enzyme, and 3) differs from the 27- to 29-kDa hepatic 5'-MD in the carboxy-terminal portion of the molecule. The 31-kDa protein labeled with [I-125]BrAcT3 may be the substrate-binding subunit of 5-MD. The presence of a similar, albeit weak, affinity-labeled band in some inactive tissues may signify that multiple subunits are required for 5-MD activity.