A highly sensitive, specific, and reproducible RIA has been developed to measure rat type I iodothyronine 5'-monodeiodinase (5'-MD). A 16-amino acid peptide (LAP-744) corresponding to a portion of the carboxy-terminal region of the rat liver 5'-MD, as predicted from its cDNA, was synthesized, and rabbits were immunized with the peptide-BSA conjugate. In a final dilution of 1:15,000, our anti-5'-MD antibody bound about 30-35% of a tracer amount of [125I]LAP-744. The detection threshold of the RIA approximated 0.08 pmol LAP-744 or an equivalent amount of 0.08 pmol 5'-MD. Rat liver and kidney microsomes produced dose-response curves that were essentially parallel to that of LAP-744. No inhibition of binding of [125I]LAP-744 to antibody was produced by 0.3 mg or less rat microsomal proteins from testes, heart, brain, muscle, spleen, intestine, lung, placenta, or fetal liver. Recovery of nonradioactive LAP-744 added to spleen microsomes averaged 103%. The coefficient of variation averaged 4% within an assay and 11% between assays. In 16 normal rats studied, the mean (+/- SD) 5'-MD content was 2.4 +/- 0.22 pmol/mg protein in liver microsomes and 2.5 +/- 0.27 pmol/mg protein in kidney microsomes. Fasting of the rat for 2-4 days was associated with a significant reduction in both the activity and the content of the 5'-MD in liver and kidney. Hypothyroidism was also associated with a significant decrease in the activity and content of 5'-MD in both tissues. Significant opposite changes were observed in these parameters in hyperthyroidism. Treatment of the rat with sodium ipodate for 3 days was associated with a significant decrease in both the activity and the content of 5'-MD in liver and kidney. A similar treatment of the rat with propylthiouracil induced a clear reduction in the activity of 5'-MD in liver and kidney, but the content of the enzyme was significantly increased in both tissues. Rats treated with aurothioglucose for 3 days exhibited a significant decrease in 5'-MD activity in liver and kidney microsomes, whereas the tissue content of 5'-MD was not affected. A similar treatment of the rat with methimazole had no significant effect on either the activity or the content of 5'-MD.(ABSTRACT TRUNCATED AT 400 WORDS)
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