Microheterogeneous forms of radioiodinated bovine thyrotropin: discrimination of different receptor-active components by gel permeation chromatography.
The products of the radioiodination and subsequent receptor adsorption of bovine TSH (bTSH) radiolabeled by the lactoperoxidase method have been further investigated. After receptor adsorption, [125I]bTSH was resolved by gel permeation chromatography on Sephadex G-100 (superfine) under low ionic strength conditions into three peaks of radioactivity (tracers 2a, 2b, and 2c, respectively). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions demonstrated that each tracer component was radiolabeled on both the alpha- and beta-subunits. Analysis of the three tracers by TSH radioreceptor assay (under different radioreceptor assay conditions) showed that tracers 2b and 2c exhibited saturable rebinding to crude thyroid membranes containing functional TSH receptors. However, tracer 2c exhibited a maximum binding 2-fold greater than tracer 2b. This difference has been attributed to the abundance of an apparently low affinity binding component in tracer 2c. Rechromatography of tracers 2b and 2c on Sephadex G-100 (superfine) under high ionic strength conditions yielded tracer profiles that were coincident, demonstrating that the initial separation under low ionic strength conditions was not based on differences in molecular volume. The data indicate that radioiodination of highly purified bTSH yields multiple tracer components. Further, receptor adsorption, commonly used to purify freshly iodinated bTSH before radioreceptor assay, purifies at least two species of receptor-active [125I] bTSH.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes / Q000378:metabolism; D007553:Isotope Labeling; D007784:Lactoperoxidase; D009994:Osmolar Concentration; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011989:Receptors, Thyrotropin; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D007784:Lactoperoxidase