Simultaneous isolation of endogenous digoxin-like immunoreactive factor, ouabain-like factor, and deglycosylated congeners from mammalian tissues.
Simultaneous isolation of endogenous digoxin-like immunoreactive factor, ouabain-like factor, and deglycosylated congeners from mammalian tissues.
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从哺乳动物组织中同时分离内源性地高辛样免疫反应因子、哇巴因样因子和去糖基化同系物。
DOI：
10.1006/abbi.1996.0160
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发表时间：
1996
影响因子：
3.9
通讯作者：
ValdesJr,R
中科院分区：
文献类型：
--
作者：
Qazzaz,HM;ValdesJr,R
DLIF（地高辛样免疫反应因子）和 OLF（哇巴因样因子）是内源性类固醇样配体（分别约为 781 和 595 Da），其分子和结构特性与植物来源的强心苷、地高辛和哇巴因相似。我们开发了一种具有足够广泛的萃取溶解度的纯化方法，可以分离极性涵盖哇巴因和地高辛的化合物。该技术提供了一种快速、可靠且有效的方法，用于同时分离 DLIF、OLF 和几种天然存在的去糖基化同源物，包括 DLIF 的三种去糖基化物种（DLIF-genin、DLIF-mono 和 DLIF-bis）和 OLF 的一种去糖基化物种（OLF-genin）。使用酸萃取、C-18 反相 HPLC 色谱法和使用两种抗体（一种针对地高辛，另一种针对哇巴因）进行信号检测来实现分离。 DLIF 和 OLF 的平均提取效率分别为每克肾上腺皮质 400 pmol 地高辛当量（范围 300-500）和 42 pmol 哇巴因当量（范围 37-50）。 DLIF 的相对摩尔免疫反应性比地高辛低 103 倍，而 OLF 的相对摩尔免疫反应性与哇巴因相比是一致的，这表明 OLF 在结构上与哇巴因更相似，而不是 DLIF 与地高辛相似。令人感兴趣的是与地高辛和哇巴因抗体发生反应的化合物的存在。这种独特的免疫反应物质可能与地高辛和哇巴因具有结构相似性，因此可能代表 DLIF 和 OLF 之间的代谢联系。
DLIF (digoxin-like immunoreactive factor) and OLF (ouabain-like factor) are endogenous steroid-like ligands (approximately 781 and 595 Da, respectively) with molecular and structural properties similar to the plant-derived cardiac glycosides, digoxin and ouabain. We developed a purification method with a sufficiently wide range of extraction solubility to separate compounds with polarities spanning those of ouabain and digoxin. This technique provides a rapid, reliable, and efficient method for simultaneously isolating DLIF, OLF, and several naturally existing deglycosylated congeners, including three deglycosylated species of DLIF (DLIF-genin, DLIF-mono, and DLIF-bis) and one deglycosylated species of OLF (OLF-genin). Separation is achieved using acid extraction, C-18 reverse-phase HPLC chromatography, and signal detection using two antibodies, one specific for digoxin and one for ouabain. The average extraction efficiency is 400 pmol digoxin equivalent (range 300–500) and 42 pmol ouabain equivalent (range 37–50) per gram of adrenal cortex for DLIF and OLF, respectively. The relative molar immunoreactivity of DLIF is 103-fold less than that of digoxin, whereas that of OLF is unity compared to ouabain, suggesting that OLF is structurally more similar to ouabain than DLIF is to digoxin. Of interest is the presence of a compound reacting with both digoxin and ouabain antibodies. This unique immunoreactive species is likely to have structural similarity to both digoxin and ouabain and thus may represent a metabolic link between DLIF and OLF.