Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

Journal Article · Sun Apr 15 00:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:7118071

Koehrle, J R; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D ^[1]

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1. Abteilung Klinische Endokrinologie, Hannover (Germany, F.R.)

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc(125I)L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc(125I)T4 yielded higher affinity label incorporation than BrAc(125I)T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney.

• OSTI ID: 7118071
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:11; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AMINO ACIDS
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
GLANDS
HORMONES
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KIDNEYS
LABELLING
LIGANDS
LIVER
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MICROSOMES
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC IODINE COMPOUNDS
ORGANOIDS
ORGANS
PEPTIDE HORMONES
PEPTIDE HYDROLASES
PROTEINS
RADIOISOTOPES
RATS
RIBOSOMES
RODENTS
TEMPERATURE DEPENDENCE
THYROID HORMONES
THYROXINE
TIME DEPENDENCE
TRIIODOTHYRONINE
VERTEBRATES