Horiuchi R, Yamauchi K, Hayashi H, Koya S, Takeuchi Y, Kato K, Kobayashi M, Takikawa H
Institute of Endocrinology, Gunma University, Maebashi, Japan.
Eur J Biochem. 1989 Aug 15;183(3):529-38. doi: 10.1111/j.1432-1033.1989.tb21081.x.
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.
牛肉肝细胞膜被证明含有不同种类的3,5,3'-三碘-L-甲状腺原氨酸结合蛋白，包括55 kDa蛋白，通过用N-溴乙酰基-3,5,3'-[125I]三碘-L-甲状腺原氨酸进行亲和标记，该蛋白在许多细胞中都被报道具有这种活性。为了表征这些结合蛋白的分子特征，从富含质膜的牛肉肝膜组分中纯化出55 kDa蛋白。该蛋白用0.5%的Chaps溶解，并通过凝胶过滤、羟基磷灰石和Mono Q阴离子交换柱色谱法进行纯化。通过反相HPLC和SDS/PAGE确认了纯度。因此，膜组分中0.4%的总蛋白被回收为55 kDa蛋白。该蛋白四分之一的氨基酸组成为谷氨酰胺（14.6%）加天冬酰胺（11.7%），其pI为4.5。纯化后的蛋白具有三碘甲状腺原氨酸结合活性，Kd为57 nM，这与膜的高亲和力结合位点相似。抗（55 kDa蛋白）血清能特异性识别牛肉、大鼠和人类细胞中的55 kDa蛋白。抗（55 kDa蛋白）血清的免疫球蛋白G组分抑制三碘甲状腺原氨酸与牛肉肝膜组分的结合。通过使核糖核酸酶复性测定，纯化后的蛋白还表现出蛋白二硫键异构酶（EC 5.3.4.1）的活性。这些数据有力地表明，具有三碘甲状腺原氨酸结合活性和蛋白二硫键异构酶活性的多功能55 kDa蛋白，据报道它还是脯氨酰-4-羟化酶的β亚基、寡糖基转移酶的糖基化位点结合蛋白和碘甲状腺原氨酸5'-单脱碘酶，可能在三碘甲状腺原氨酸对靶细胞的作用中起重要作用。
