Large scale purification of the nuclear thyroid hormone receptor from rat liver and sequence-specific binding of the receptor to DNA.
Methodology is reported for extracting thyroid hormone receptors from rat liver nuclei and for purifying these such that certain receptor properties can be examined. The extraction technique resulted in 1700 pmol of receptor/2 kg of liver and bypasses centrifugation in dense sucrose. The receptor was then purified by sequential heparin-Sepharose, DEAE-Sepharose, and phospho-Ultrogel chromatography and size exclusion and hydrophobic interaction high performance liquid chromatography. These steps yielded 23-35 micrograms of receptor at 0.7-1.5% purity from two 2-kg liver preparations. The cross-linkers disuccinimidyl suberate and N-succinimidyl-6-(4-azido-2-nitrophenylamino)hexanoate were employed to covalently attach 125I-labeled 3,5,3'-triiodo-L-thyronine (T3) to the purified receptor. Autoradiography after denaturing polyacrylamide gel electrophoresis revealed major 49,000 Mr and minor 58,000 Mr specific T3-binding proteins. The purified receptors exhibited high affinity (Kd = 100 pM) single site T3-binding activity. Because of the high affinity and specificity of [125I]T3 for the receptor, it was possible to uniquely identify the receptor containing DNA-protein complexes in a gel retardation assay and thus directly demonstrate for the first time that the receptor can specifically recognize sequences in the 5'-flanking DNA of the rat growth hormone gene. [125I]T3-labeled receptor migrated at the same position as the major gel-retarded 32P-labeled DNA band. Specific DNA competed for the binding much more strongly than nonspecific DNA. Thus, the purification procedure results in relatively large quantities of receptor at a purity sufficient for detecting and studying a number of its properties including specific DNA binding activity.
Apriletti
J W
JW
Metabolic Research Unit, University of California, San Francisco 94143.
Baxter
J D
JD
Lavin
T N
TN
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification* / Q000378:metabolism
D011988:Receptors, Thyroid Hormone; D004247:DNA