Purification and characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line.
A membrane-associated binding protein for 3,3',5-triiodo-L-thyronine (T3) was purified to apparent homogeneity from A431 human epidermoid carcinoma cells. A431 cells were specifically labeled with the N-bromoacetyl derivative of T3 labeled with 125I at the 3' position (BrAc[125I]T3) and were extracted with 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized BrAc[125I]T3-labeled protein was successively purified by chromatography on Sephadex G-200 and QAE-Sephadex followed by NaDod-SO4/PAGE. Approximately 0.2 mg of purified protein was obtained from 2.5 X 10(9) cells, which represents a 3000-fold purification. The membrane-associated T3 binding protein is an acidic protein with a pI of 5.1 and an apparent molecular mass of 55,000 daltons determined by NaDodSO4/PAGE. Polyclonal antibodies against the 55-kDa protein were prepared and used in indirect immunofluorescence to show that the 55-kDa protein was mainly found in the nuclear envelope and endoplasmic reticulum.
D002294:Carcinoma, Squamous Cell / Q000032:analysis*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002793:Cholic Acids; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D011988:Receptors, Thyroid Hormone; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D002793:Cholic Acids; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate