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′‐[ 125 I]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 K d 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 β 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.