A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I.
A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I.
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发表时间：
2001-05
影响因子：
6.5
通讯作者：
W. Safi;J. N. Maiorano;W. Davidson
中科院分区：
文献类型：
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作者：
W. Safi;J. N. Maiorano;W. Davidson
最近的研究表明，载脂蛋白（apo）A-I的某些脂质贫乏形式可能是特别重要的，在促进胆固醇释放超负荷细胞在周边。然而，详细了解这些物种的生理相关性一直阻碍了测量它们的困难。作为对这些形式的apoA-I的快速测定的搜索的一部分，我们已经观察到蛋白酶肠肽酶可以特异性地切割人无脂质apoA-I，但不能切割其脂质结合形式。肠肽酶在氨基酸188处的单个位点切割无脂质apoA-I，产生22 kDa的N-末端片段。然而，当存在于直径小至6 nm的重组高密度脂蛋白（rHDL）颗粒或人HDL（3）颗粒中时，即使在极高的酶-蛋白比和延长的反应时间下，apoA-I对肠肽酶也不敏感。我们利用这一观察结果开发了一种在体外系统中测量贫脂apoA-I的测定方法。使用光密度法从十二烷基硫酸钠聚丙烯酰胺凝胶生成标准曲线，以确定用肠肽酶处理的未知样品中N-末端蛋白水解片段的量。该系统可以准确地定量从rHDL颗粒和人HDL（3）中置换的apoA-I和纯化的apoA-II。在此基础上，提出了“无脂”、“贫脂”和“脂结合”apoA-I的命名系统。所报道的方法区分形式的apoA-I的构象参数没有预先分离的物种。该方法简便、价廉，有助于了解血浆HDL中有利于apoA-I解离的特征。
Recent studies indicate that certain lipid-poor forms of apolipoprotein (apo)A-I may be particularly important in promoting cholesterol release from overburdened cells in the periphery. However, a detailed understanding of the physiological relevance of these species has been hampered by the difficulty in measuring them. As part of a search for a rapid assay for these forms of apoA-I, we have observed that the protease enteropeptidase can specifically cleave human lipid-free apoA-I but not its lipid-bound form. Enteropeptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resulting in an N-terminal fragment of 22 kDa. However, apoA-I was not susceptible to enteropeptidase when present in reconstituted high-density lipoprotein (rHDL) particles as small as 6 nm in diameter or in human HDL(3) particles, even at extremely high enzyme-to-protein ratios and extended reaction times. We capitalized on this observation to develop an assay for the measurement of lipid-poor apoA-I in in vitro systems. Densitometry was used to generate a standard curve from sodium dodecyl sulfate polyacrylamide gels to determine the amounts of the N-terminal proteolytic fragment in unknown samples treated with enteropeptidase. This system could accurately quantify apoA-I that had been displaced from rHDL particles and human HDL(3) with purified apoA-II. On the basis of the results, a system of nomenclature is proposed for "lipid-free," "lipid-poor," and "lipid bound" apoA-I. The reported method distinguishes forms of apoA-I by a conformational parameter without previous separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorable for the dissociation of apoA-I.