Tang W X, Bazaraa H M, Magiera H, Cooke N E, Haddad J G
Department of Medicine, University of Pennsylvania, School of Medicine, 415 Curie Boulevard, Philadelphia, Pennsylvania, 19104-6149, USA.
Anal Biochem. 1996 Jun 1;237(2):245-51. doi: 10.1006/abio.1996.0236.
Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.
血清维生素D结合蛋白（DBP，也称为Gc球蛋白）是一种多功能蛋白，能够结合维生素D代谢产物和肌动蛋白。通过聚丙烯酰胺凝胶电泳然后染色进行分析时，可以看到DBP。此前，确认其身份需要用特异性抗DBP抗血清进行免疫沉淀，或者用放射性维生素D固醇占据该蛋白。我们的研究表明，将G-肌动蛋白与哺乳动物血清预孵育，在天然凝胶电泳上会产生可辨别的DBP蛋白条带迁移。在孵育体系中加入DNaseI（一种具有强烈肌动蛋白结合位点的33 kDa细胞内蛋白），会导致DBP-肌动蛋白复合物超迁移至凝胶中更偏向阴极的区域。在与人、大鼠和小鼠血清孵育后，出现了与肌动蛋白加DNaseI超迁移相同的肌动蛋白迁移。在相同条件下，每种复合物的迁移与纯化的DBP迁移相关。通过蛋白质印迹法以及通过25-OH[3H]D3结合检测DBP，证实超迁移条带中含有DBP。给活体小鼠静脉注射G-肌动蛋白后，随着未结合的DBP条带强度降低，在天然凝胶上可以检测到血清DBP-肌动蛋白复合物。这种简单的直接染色技术似乎适用于识别人类群体中的DBP/Gc表型，以及在动物模型或人类疾病中对体内血浆肌动蛋白清除系统进行半定量监测。
