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. T he present study was undertaken to characterize rat placental type III iodothyronine 5-MD and to compare it with 5'-MD. Solubilized rat plac ental microsomes were delipidated by carboxymethyl cellulose-Sephadex chromatography. Phospholipids and proteins were recovered in two disti nct peaks, which did not show 5-MD activity. 5-MD activity was recover ed fully, however, by combining the two components (phospholipids and protein) and partially after the addition of exogenous phospholipids t o protein. Tissue selenoproteins were labeled by injection of radioact ive 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 electropho resis, followed by autoradiography. No specific seleno-labeled protein s were evident in the microsomes of placenta or maternal or fetal brai n. A 27- to 29-kilodalton (kDa) band previously suggested to be 5'-MD was observed, however, in maternal liver and kidney microsomes. Auroth ioglucose inhibited rat placental 5-MD, but the dose required for 50% inhibition was over 50-fold greater than that for Se-containing hepati c 5'-MD (430 vs. 8 nM). The mechanism of the inhibition was noncompeti tive for 5-MD, whereas it was competitive for 5'-MD. A synthetic pepti de 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 ba nds were observed in 5-MD-rich placental or fetal brain microsomes. Br omoacetyl T3 (BrAcT3) was a potent inhibitor of rat placental 5-MD. Af finity labeling of solubilized rat placental microsomes with [I-125]Br AcT3 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 wa s enhanced by 10 mM dithiothreitol, inhibited 60% by 150-mu-M T3, and prevented by 40-mu-M aurothioglucose. A dominant affinity-labeled 31-k Da band was also observed in fetal brain microsomes. Some tissues with out 5-MD activity (testes and spleen) also showed weak binding. We con clude that rat placental 5-MD 1) requires phospholipids for enzymatic activity, 2) is not a seleno-containing enzyme, and 3) differs from th e 27- to 29-kDa hepatic 5'-MD in the carboxy-terminal portion of the m olecule. The 31-kDa protein labeled with [I-125]BrAcT3 may be the subs trate-binding subunit of 5-MD. The presence of a similar, albeit weak, affinity-labeled band in some inactive tissues may signify that multi ple subunits are required for 5-MD activity.