A cellular 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line. Purification and characterization.
A cellular binding protein for 3,3',5-triiodo-L-thyronine (T3) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from A431 human epidermoid carcinoma cells. The binding activity is T3 specific. Analysis of the equilibrium binding data indicated that the binding protein has one class of binding sites for T3 with a Kd of (17 +/- 3) nM and Bmax of (1.8 +/- 0.6) pmol/50 micrograms of protein. The pH optimum for binding is 6.8. The T3 binding protein elutes from Sephadex G-200 in an included peak which has a Stokes radius of 40 A and sediments on glycerol gradients at 3.7 S. By affinity labeling with [3,5-125I]thyroxine a protein with a molecular weight of 58,000 was specifically labeled. Its isoelectric point was determined to be 7.1, which is different from the reported pIs of other thyroid hormone binding proteins. p58 was successively purified to apparent homogeneity by chromatography on Sephadex G-200, QAE-Sephadex, SP-Sephadex, and hydroxylapatite. Approximately 50 micrograms of purified protein was obtained from 2.5 X 10(9) cells with a yield of 1.1%. The purified protein retains its binding activity. The specific binding activity is enriched by approximately 1000-fold. With the availability of a purified protein with T3 binding activity, it becomes possible to study its cellular function.
D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D002460:Cell Line; D002793:Cholic Acids; D003902:Detergents; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D002793:Cholic Acids; D003902:Detergents; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate