A radioimmunoassay of rat type I iodothyronine 5'-monodeiodinase.
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)
Santini
F
F
Department of Medicine, University of California Center for the Health Sciences, Los Angeles 90024.
Chopra
I J
IJ
D000595:Amino Acid Sequence; D000818:Animals; D005215:Fasting; D005260:Female; D006980:Hyperthyroidism / Q000201:enzymology; D007037:Hypothyroidism / Q000201:enzymology; D007453:Iodide Peroxidase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007487:Ipodate / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000196:embryology / Q000201:enzymology; D008297:Male; D008713:Methimazole / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010446:Peptide Fragments / Q000276:immunology; D011441:Propylthiouracil / Q000494:pharmacology; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010446:Peptide Fragments; D008713:Methimazole; D011441:Propylthiouracil; D007453:Iodide Peroxidase; D007487:Ipodate