Binding of thyroid hormones to human hemoglobin and localization of the binding site.
Radiolabeled thyroid hormones were allowed to bind to erythrocyte cytosol and the complex was fractionated by Sephadex G-100 or by high-performance liquid chromatography (HPLC). On Sephadex G-100, four radioactive peaks (P1-P4) were obtained, whereas HPLC gave only three radioactive peaks (P1-P3). Chromatographic studies with human adult Hb and non-Hb cytosol protein fractions, which had been reacted with radiolabeled thyroid hormones, and immune precipitation with specific antisera for the hormones, confirmed that the first peak of Sephadex G-100 radioactivity was a mixture of Hb and non-Hb proteins, while the second peak was Hb. The third peak was free 125I and the fourth peak was unbound 125I-T3 or 125I-T4. The third peak of HPLC was confirmed to be a mixture of free 125I and unbound radiolabeled thyroid hormones. Scatchard analysis of the interaction between T4 and apo-Hb, and the alpha- and beta-chains of human Hb suggested the presence of the specific binding site(s) for the hormone. Interaction between T4 and synthesized peptides, which constitute the heme pocket of the beta-chain of Hb (beta 61-75, beta 71-85, beta 81-95), indicated that the T4 binding site of Hb resides within the heme-binding cavity. It is concluded that human erythrocyte cytosol does not contain "receptor" for thyroid hormones and cannot be a model for studying functions of cytosol "receptor" for the hormones; rather, it contains binding protein with large binding capacity, including Hb and non-Hb proteins, which possibly constitute a large reservoir for the hormone in blood.
Sakata
S
S
Third Department of Internal Medicine, Gifu University School of Medicine, Japan.
Komaki
T
T
Nakamura
S
S
Ohshima
M
M
Sagisaka
K
K
Yoshioka
N
N
Atassi
M Z
MZ
Miura
K
K
D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006418:Heme; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000138:chemical synthesis; D011233:Precipitin Tests; D011485:Protein Binding; D013974:Thyroxine / Q000737:chemistry / Q000378:metabolism*; D014284:Triiodothyronine / Q000737:chemistry / Q000378:metabolism*
D006454:Hemoglobins; D010446:Peptide Fragments; D014284:Triiodothyronine; D006418:Heme; D013974:Thyroxine