Separation of DNA binding domain from hormone and core histone binding domains by trypsin digestion of rat liver nuclear thyroid hormone receptor.
Rat liver nuclear thyroid hormone receptor was subjected to limited trypsin digestion, and the tryptic fragment of the 3,5,3'-triiodo-L-thyronine (T3)-receptor complex was characterized. Rat liver nuclear thyroid hormone receptor is an asymmetrical protein with Stokes radius of 34 A, sedimentation coefficient of 3.4 S, and molecular weight of 49,000. A globular T3-receptor complex with Stokes radius of 22 A, sedimentation coefficient of 2.8 S, and molecular weight of 26,000 was obtained by tryptic digestion. This fragment had no DNA binding activity, whereas undigested receptor showed significant DNA binding activity. Addition of undigested receptor to the tryptic fragment did not restore DNA binding activity of digested receptor, nor did mixing inhibit DNA binding activity of undigested receptor complex. Undigested receptor bound to core histones, and this activity was stronger than with other proteins tested (H1 histone, cytochrome c, and ovalbumin). The tryptic fragment of receptor maintained core histone binding activity comparable to that of undigested receptor. The tryptic fragment had affinity for T3 comparable to undigested receptor as assessed by Scatchard analysis and the same rate for dissociation of [125I]T3 from receptor. The tryptic fragment of the T3-receptor complex was more stable than undigested receptor at 43 degrees C. Digestion of receptor unoccupied by T3 caused a significantly larger loss of T3 binding capacity than did digestion of T3-occupied receptor, suggesting a protective effect of T3 on a second trypsin-sensitive site on the receptor, which, when cut, destroys T3 binding activity.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D006657:Histones; D011988:Receptors, Thyroid Hormone; D004247:DNA; D014357:Trypsin