A study of the characteristics of hepatic iodothyronine 5'-monodeiodinase in various vertebrate species.
Rat type I iodothyronine 5'-monodeiodinase (5'-MD) has recently been shown to be a selenium-containing enzyme. In the present study we compared the characteristics of the 5'-MD from liver microsomes of rat, mouse, guinea pig, man, beef, pig, sheep, and chicken. Aurothioglucose (ATG), a known potent inhibitor of selenium-containing enzymes, was a consistent, very potent inhibitor of 5'-MD activity in all species studied, with a 50% inhibitory dose in the narrow range of 5.8-12 nM. ATG was also a potent and selective inhibitor of [125I]bromoacetyl T3 affinity labeling of 5'-MD. Thus, in the species studied, only one affinity-labeled band, which was selectively displaced by gold, was identified. The mol wt of the affinity-labeled proteins in various liver microsomal preparations ranged between 28-36 kilodaltons (kDa), and the ATG concentrations necessary for the inhibition of affinity labeling of microsomes with [125I]bromoacetyl T3 were comparable to those required for inhibition of the enzyme activity in all species except the pig. The pig liver microsomes demonstrated a dominant affinity-labeled 36-kDa band, but much higher ATG concentrations (micromolar) were required for inhibition of affinity labeling. In view of the potent inhibition of pig liver 5'-MD activity by ATG, it appears unlikely that this band in the pig corresponds only to the substrate-binding site of 5'-MD, but this issue requires further study. A synthetic peptide of 16 amino acids corresponding to the carboxy-terminal portion of rat 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 and kidney microsomes and thyroid homogenate. No specific bands were observed in other adult rat tissues studied or in fetal rat liver. No specific bands were observed when Western blot studies with antibody against the carboxy-terminal portion of rat 5'-MD were performed in liver microsomes from species other than the rat. In conclusion, our studies indicate that selenium is a likely component of type I 5'-MD in all species studied. However, substantial structural differences exist between the rat type I 5'-MD and that in various other species.
Santini
F
F
Department of Medicine, University of California School of Medicine, Los Angeles 90024.
Chopra
I J
IJ
Hurd
R E
RE
Teco
G N
GN
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D006051:Aurothioglucose / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D006168:Guinea Pigs; D006801:Humans; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011441:Propylthiouracil / Q000494:pharmacology; D051381:Rats; D012643:Selenium / Q000032:analysis; D012756:Sheep; D013552:Swine; D014284:Triiodothyronine / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D014284:Triiodothyronine; D006051:Aurothioglucose; D011441:Propylthiouracil; C036219:N-bromoacetyl-3,3',5-triiodothyronine; D007453:Iodide Peroxidase; D012643:Selenium