Battiston L, Passamonti S, Macagno A, Sottocasa G L
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
Biochem Biophys Res Commun. 1998 Jun 29;247(3):687-92. doi: 10.1006/bbrc.1998.8868.
In the primary structure of bilitranslocase, currently under study in our laboratory, an aminoacid motif was identified and found to be conserved in a number of alpha-phycocyanines, ancient biliproteins present in cyanobacteria. To test the possibility that such a motif could be at least part of the binding site for bilirubin, epitope-specific antibodies were raised. The target corresponds to the sequence 65-75 of bilitranslocase and covers the central portion of the motif identified. The antibodies were shown: 1) to inhibit the electrogenic BSP transport by plasmamembrane vesicles; 2) to react with purified bilitranslocase; and 3) to identify only one protein band with electrophoretic mobility identical to bilitranslocase in Western blots of solubilised plasmamembrane vesicles. The presence of either bilirubin or nicotinate during pre-incubation with the antibodies decreases concentration-wise the inhibition kinetics. From these experiments a dissociation constant of 2.2 +/- 0.3 and 11.3 +/- 1.3 nM for bilirubin-bilitranslocase and nicotinate-bilitranslocase complexes were calculated.
在我们实验室目前正在研究的胆红素转运酶的一级结构中，鉴定出了一个氨基酸基序，并且发现它在一些α-藻蓝蛋白中是保守的，α-藻蓝蛋白是蓝细菌中存在的古老胆色素蛋白。为了测试这样一个基序至少可能是胆红素结合位点一部分的可能性，制备了表位特异性抗体。靶标对应于胆红素转运酶的65-75位序列，覆盖了所鉴定基序的中心部分。这些抗体表现出：1）抑制质膜囊泡的电致性BSP转运；2）与纯化的胆红素转运酶发生反应；3）在溶解的质膜囊泡的蛋白质免疫印迹中仅识别出一条电泳迁移率与胆红素转运酶相同的蛋白带。在与抗体预孵育期间存在胆红素或烟酸会按浓度降低抑制动力学。从这些实验中计算出胆红素-胆红素转运酶和烟酸-胆红素转运酶复合物的解离常数分别为2.2±0.3和11.3±1.3 nM。
