Okamoto K, Liu G, Yu W G, Isohashi F
Department of Biochemistry, St. Marianna University School of Medicine, Kanagawa.
J Biochem. 1994 May;115(5):862-7. doi: 10.1093/oxfordjournals.jbchem.a124431.
We previously found a novel endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor-translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. In this work, we immunized rabbits with the purified ASTP protein and characterized the antibodies with regard to titer, cross-reactivity and specificity. An IgG fraction from sera of the immunized rabbits contained specific antibodies to ASTP. The anti-ASTP IgG could precipitate the ASTP protein without the activity. Immunoblot analysis revealed a major band of 48 kDa in rat liver cytosol that migrated to the same position as the purified ASTP protein by SDS-PAGE, and an additional minor band of about 50 kDa. Monospecific antibodies purified from the IgG fraction using the antigen (the purified 48-kDa ASTP protein) immobilized on a polyvinylidene difluoride membrane also reacted with both the 48-kDa ASTP protein and the 50-kDa protein in rat liver cytosol, suggesting that this 50-kDa protein is immunologically related to the 48-kDa ASTP protein. Densitometric quantification of immunoblots demonstrated that the rat kidney cytosol contained ASTP protein at a concentration of about 20% of that of liver cytosol. Other tissues such as brain, skeletal muscle, heart, and lung, contained neither the ASTP protein nor the activity.
我们之前在大鼠肝脏胞质溶胶中发现了一种新的内源性因子，命名为ATP刺激的糖皮质激素受体易位启动子（ASTP），它在ATP存在的情况下增加了活化的糖皮质激素受体与细胞核的结合。在这项研究中，我们用纯化的ASTP蛋白免疫兔子，并对抗体的效价、交叉反应性和特异性进行了表征。免疫兔子血清中的IgG组分含有针对ASTP的特异性抗体。抗ASTP IgG可以沉淀无活性的ASTP蛋白。免疫印迹分析显示，大鼠肝脏胞质溶胶中有一条48 kDa的主要条带，通过SDS-PAGE迁移到与纯化的ASTP蛋白相同的位置，还有一条约50 kDa的次要条带。使用固定在聚偏二氟乙烯膜上的抗原（纯化的48 kDa ASTP蛋白）从IgG组分中纯化的单特异性抗体也与大鼠肝脏胞质溶胶中的48 kDa ASTP蛋白和50 kDa蛋白发生反应，表明这种50 kDa蛋白与48 kDa ASTP蛋白在免疫上相关。免疫印迹的光密度定量分析表明，大鼠肾脏胞质溶胶中ASTP蛋白的浓度约为肝脏胞质溶胶的20%。其他组织如脑、骨骼肌、心脏和肺，既不含ASTP蛋白也无该活性。
