Isolation, stabilization, and molecular weight estimation of thyroid hormone receptors of tadpole and chick embryo erythrocytes.
Typical procedures for the isolation of triiodothyronine (T3) receptors from mammalian nuclei involve extraction of nuclei with buffers containing divalent cations and 0.40M KCl. However, when applied to tadpole erythrocyte (RBC) nuclei, this method gave low yields of relatively unstable T3 receptors. The use of EDTA (10 mM) and 0.4M KCl in a sucrose-Tris buffer resulted in the extraction of 90% of the specifically bound [125I]-T3 from RBC nuclei. It was also found that 5 mM thiol reagent (DTT, GSH, or beta-mercaptoethanol) was required for maximal stability of the receptor. Fractionation of labeled RBC nuclear extracts on a Sephadex G-100 column yielded only one peak of specific T3 binding activity. The T3 receptor peak eluted at the same position as bovine serum albumin (BSA), with an estimated mol wt of 68 kDa. Specific T3 binding activity was destroyed by protease digestion but not by DNAse or RNAse. Scatchard analysis of the fractions from the receptor peak supported the existence of one class of T3 binding sites, with an estimated Kd (about 7 pM) comparable to the Kd reported for the intact RBC. Using the same methods, T3 receptors from the nuclei of chick embryo RBCs were also isolated, again with a Kd (7 pM) similar to that for the intact RBC. The chick receptor also eluted from the Sephadex G-100 column at the same position as BSA. The estimated mol wt of the T3 receptors from both sources is comparable to those reported for T3 receptors from other sources. The results show that T3 receptors derived from both tadpole and chick RBC nuclei could be isolated in a soluble and stable form with no apparent change in Kd.
Dasmahapatra
A K
AK
Department of Chemistry, Florida State University, Tallahassee 32306.
Thomas
C R
CR
Frieden
E
E
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002457:Cell Extracts; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D002850:Chromatography, Gel; D002918:Chymotrypsin; D004912:Erythrocytes / Q000737:chemistry* / Q000648:ultrastructure; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007814:Larva / Q000378:metabolism; D008970:Molecular Weight; D011892:Rana catesbeiana; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014284:Triiodothyronine / Q000097:blood*; D014357:Trypsin
D002457:Cell Extracts; D007457:Iodine Radioisotopes; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D002918:Chymotrypsin; D014357:Trypsin