The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat.
Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.
Leonard
J L
JL
Molecular Endocrinology Laboratories, University of Massachusetts Medical School, Worcester 01655, USA. jack.leonard@banyan.ummed.edu
Leonard
D M
DM
Safran
M
M
Wu
R
R
Zapp
M L
ML
Farwell
A P
AP
D002001:Adipose Tissue, Brown / Q000737:chemistry; D000818:Animals; D001253:Astrocytes / Q000201:enzymology; D001254:Astrocytoma; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D015870:Gene Expression; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D007453:Iodide Peroxidase / Q000032:analysis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis; D000097564:Iodothyronine Deiodinase Type II
D003994:Bucladesine; D018076:DNA, Complementary; D007453:Iodide Peroxidase; D012333:RNA, Messenger; D000097564:Iodothyronine Deiodinase Type II