Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane.
Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.
Institute of Endocrinology, Gunma University, Maebashi, Japan.
R
R
Horiuchi
K
K
Yamauchi
H
H
Hayashi
S
S
Koya
Y
Y
Takeuchi
K
K
Kato
M
M
Kobayashi
H
H
Takikawa
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007535:Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D019704:Protein Disulfide-Isomerases; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
D000596:Amino Acids; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D007535:Isomerases; D019704:Protein Disulfide-Isomerases