Ye J M, Key C J, Wolfe J L
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis 38163, USA.
Biochem Biophys Res Commun. 1996 Jun 5;223(1):153-9. doi: 10.1006/bbrc.1996.0861.
Protein disulfide isomerase has broad specificity in the catalysis of the formation and rearrangement of native disulfide bonds in proteins. This enzyme has two independent thioredoxin-like active sites (-CGHC-) and a peptide binding site. However, the mechanisms involving the catalytic processes are not clearly understood. It was reported that the enzyme associates with scrambled pancreatic ribonuclease A in vitro, and with misfolded human lysozyme in vivo. In the present study, recombinant human interleukin 2 has been chosen to probe the reaction intermediate in the reaction with the enzyme. We have identified and characterized a covalent associate formed in vitro by SDS-PAGE and Western blot analysis. This associate has a molecular weight of 71-72 kDa, the approximate sum of the molecular weights of the enzyme and the substrate. Western blot analysis confirmed that it formed via an intermolecular disulfide bond. Upon treatment with 2-mercaptoethanol, this bond was cleaved.
蛋白质二硫键异构酶在催化蛋白质中天然二硫键的形成和重排方面具有广泛的特异性。这种酶有两个独立的硫氧还蛋白样活性位点（-CGHC-）和一个肽结合位点。然而，涉及催化过程的机制尚不清楚。据报道，该酶在体外与混乱的胰腺核糖核酸酶A结合，在体内与错误折叠的人溶菌酶结合。在本研究中，选择重组人白细胞介素2来探测与该酶反应中的反应中间体。我们通过SDS-PAGE和蛋白质印迹分析鉴定并表征了在体外形成的共价结合物。这种结合物的分子量为71 - 72 kDa，大约是酶和底物分子量之和。蛋白质印迹分析证实它是通过分子间二硫键形成的。用2-巯基乙醇处理后，该键被裂解。
