Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin.
In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D008099:Liver / Q000032:analysis; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D011988:Receptors, Thyroid Hormone; D012995:Solubility; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D002843:Chromatin; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D013974:Thyroxine