Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine.
Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS)
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D008027:Light; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone; D013974:Thyroxine / Q000378:metabolism* / Q000528:radiation effects
D000345:Affinity Labels; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D013974:Thyroxine