Purification and characterization of rat liver nuclear thyroid hormone receptors

Journal Article · Fri May 01 04:00:00 EDT 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.10.3420· OSTI ID:5512510

Ichikawa, K; DeGroot, L J

Nuclear thyroid hormone receptor hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, the authors reproducibly obtained 6.4-14.7 ..mu..g of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity, and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO/sub 4//polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with (/sup 125/I)thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported.

• Research Organization: Univ. of Chicago, IL
• OSTI ID: 5512510
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:10; ISSN PNASA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
GLANDS
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIVER
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC IODINE COMPOUNDS
ORGANS
PEPTIDE HORMONES
PROTEINS
PURIFICATION
RADIOISOTOPES
RADIORECEPTOR ASSAY
RATS
RECEPTORS
RODENTS
THYROID HORMONES
THYROXINE
TRACER TECHNIQUES
TRIIODOTHYRONINE
VERTEBRATES