Ouabain-binding protein(s) from human plasma.
Ouabain-binding protein(s) from human plasma.
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来自人血浆的哇巴因结合蛋白。
DOI：
10.1161/01.hyp.0000027134.14160.1d
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
HaupertJr,GarnerT
中科院分区：
文献类型：
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作者：
Parhami-Seren,Behnaz;Haberly,Richard;Margolies,MichaelN;HaupertJr,GarnerT
哺乳动物Na+，K+-ATP酶上强心苷的结合位点的保守性以及Na+泵在哺乳动物细胞生理学中的重要性刺激了对这些植物化合物的哺乳动物类似物的研究。从大脑和血液中分离出的一种候选物质似乎是哇巴因本身或一种密切相关的异构体，即哇巴因样化合物。对循环形式知之甚少。由于人类类固醇激素与载体蛋白一起循环，我们制备了哇巴因特异性单克隆抗体（mAb 1-10），并用它来探测正常人血浆中的哇巴因-蛋白载体复合物。哇巴因样生物活性与80，50和25 kDa的蛋白质条带分离。这些蛋白质似乎是人免疫球蛋白或免疫球蛋白样，因为它们被抗人免疫球蛋白抗体识别，但不被抗小鼠免疫球蛋白抗体识别。含蛋白质的级分抑制mAb 1-10与固定化哇巴因的结合，并且在蛋白A上进一步纯化后，免疫球蛋白样蛋白以200至600 nmol/L的IC 50结合放射性哇巴因，但以低100倍的亲和力结合地高辛，表明对哇巴因或其异构体具有特异性。在C18上纯化后的活性蛋白组分抑制人红细胞中的Na+泵活性（IC 50 <4 nmol/L，哇巴因当量），并且该色谱似乎将哇巴因样化合物从免疫球蛋白中解离。这些免疫球蛋白样分子可能代表免疫球蛋白的一个亚组（≤总蛋白A免疫球蛋白的0.5%），在体内调节人Na+，K+-ATP酶中作为哇巴因样化合物的储库和递送系统。
Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18inhibit Na+pump activity in human erythrocytes (IC50≈4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (≤0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.