Specific detection of type III iodothyronine deiodinase protein in chicken cerebellar purkinje cells.
Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T(3) concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight ( approximately 30 kDa) could be detected in both tissues. Using a full-length (32)P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.
Verhoelst
C H J
CH
Laboratory of Comparative Endocrinology, K. U. Leuven, Zoological Institute, Belgium. carla.verhoelst@bio.kuleuven.ac.be
Vandenborne
K
K
Severi
T
T
Bakker
O
O
Zandieh Doulabi
B
B
Leonard
J L
JL
Kühn
E R
ER
van der Geyten
S
S
Darras
V M
VM
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D002642:Chick Embryo; D002645:Chickens / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D007453:Iodide Peroxidase / Q000096:biosynthesis* / Q000235:genetics; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D011689:Purkinje Cells / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
D018076:DNA, Complementary; D012333:RNA, Messenger; C117274:iodothyronine deiodinase type III; D007453:Iodide Peroxidase