Solubilization and purification of a membrane-associated 3,3',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.
3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.
Angel
R C
RC
Instituto de Quĭmica Biológica Dr. Bernabé Bloj, Facultad de Bioquímica, INSIBIO/UNT/CONICET, San Miguel de Tucumán, Argentina.
Botta
J A
JA
Morero
R D
RD
Farias
R N
RN
D000818:Animals; D002352:Carrier Proteins / Q000097:blood* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism*; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins