IDENTIFICATION OF THE LIPID BINDING SITE OF THE PHOSPHATIDYLCHOLINE EXCHANGE PROTEIN WITH A PHOTOSENSITIVE NITRENE AND CARBENE PRECURSOR OF PHOSPHATIDYLCHOLINE
IDENTIFICATION OF THE LIPID BINDING SITE OF THE PHOSPHATIDYLCHOLINE EXCHANGE PROTEIN WITH A PHOTOSENSITIVE NITRENE AND CARBENE PRECURSOR OF PHOSPHATIDYLCHOLINE
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磷脂酰胆碱光敏氮烯和卡宾前体对磷脂酰胆碱交换蛋白脂质结合位点的鉴定
DOI：
10.1111/j.1749-6632.1980.tb21304.x
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发表时间：
1980
影响因子：
5.2
通讯作者：
H. Khorana
中科院分区：
文献类型：
--
作者：
K. Wirtz;P. Moonen;L. Deenen;R. Radhakrishnan;H. Khorana
脂质-蛋白质结构如生物膜和血清脂蛋白在很大程度上从水介质的独特性质获得其稳定性和同一性。这些结构具有共同的非极性核心，但其特征在于化学上不同的脂质和蛋白质的异质混合物。为了理解这些结构的功能模式，需要深入了解脂质和蛋白质的空间组织。具体来说，这需要识别的内在蛋白质，描绘的蛋白质片段嵌入在非极性核心，并与这些疏水蛋白质片段接触的脂质的分析。在试图通过标记双层内的内在膜蛋白来阐明这种结构上的难题时，Gitler和同事3-G开发了亲脂性光敏试剂，其溶解到天然膜的非极性核心中。在肌浆网膜和红细胞中，使用1 -叠氮0 -5-[~1]碘萘已经证明，光生氮烯衍生物与内在蛋白共价偶联。在含有（Na+，K+）ATP酶的膜中，这种氮烯衍生物的偶联主要发生在这种钠泵蛋白的膜包埋片段上。5在红细胞中，大约一半的偶联产物由胰蛋白酶不溶性，血型糖蛋白的跨膜片段。O 1-叠氮基0 -4-碘-[~H]的掺入苯进入人红细胞膜的研究证实，光生氮宾与血型糖蛋白的膜内区域共价偶联。'?这些研究表明，亲脂性的，原位生成的芳基氮烯是有用的工具，以确定内在的膜蛋白和它们的部分与脂质双层接触。然而，Bayley和Kn ~ wles反对使用芳基氮烯，因为芳基氮烯相对较长的寿命和亲电子特性可能会阻止膜内蛋白质片段的所需无差别标记。这些缺点可以通过使用更反应性的卡宾来克服，所述卡宾是从作为亲脂性标记试剂的二氮杂环丙烷光化学衍生的。10与苯基氮宾相反，溶解到磷脂囊泡的双层中的苯基二氮杂环丙烷和金刚烷基二氮杂环丙烷的光生卡宾具有足够的反应性以插入饱和脂肪酸的碳氢键中。lo
Lipid-protein structures like biological membranes and serum lipoproteins derive their stability and identity to a large extent from the unique properties of the aqueous medium.lV2 These structures have an apolar core in common, yet are characterized by a heterogeneous mixture of chemically distinct lipids and proteins. To understand the mode by which these structures function, insight into the spatial organization of lipids and proteins is required. Specifically, this demands the identification of the intrinsic proteins, the delineation of the protein segments that are embedded in the apolar core, and the analysis of the lipids in contact with these hydrophobic protein segments. In an attempt to elucidate this architectural puzzle by labeling the intrinsic membrane proteins from within the bilayer, Gitler and coworkers 3-G developed lipophilic photosensitive reagents that dissolve into the apolar core of natural membranes. In sarcoplasmic reticulum membranes and erythrocytes, use of 1 -azid0-5-[~~~1]iodonaphthalene has demonstrated that the photogenerated nitrene derivative coupled covalently to the intrinsic protein^.^ In membranes containing (Na+,K+) ATPase, coupling of this nitrene derivative occurred predominantly to a membrane-embedded fragment of this sodium pump protein.5 In erythrocytes approximately half of the coupling products consisted of the trypsin-insoluble, trans-membrane segment of glycophorin.O Incorporation of l-azid0-4-iodo-[~H]benzene into human erythrocyte membranes has confirmed that the photogenerated nitrene covalently couples to the intramembranous region of glycophorin.'? fi These studies suggest that the lipophilic, in situ generated arylnitrenes are useful tools to identify the intrinsic membrane proteins and their segments in contact with the lipid bilayer. Bayley and K n ~ w l e s , ~ however, have argued against the use of arylnitrenes whose relatively long lifetime and electrophilic character may prevent the desired indiscriminate labeling of the intramembranous protein segments. These disadvantages can be overcome by the use of the more reactive carbenes, derived photochemically from diazirines as lipophilic labeling reagents.l0 In contrast to phenylnitrene, the photogenerated carbenes of phenyldiazirine and adamantyl-diazirine dissolved into the bilayer of phospholipid vesicles, are sufficiently reactive to insert into the carbonhydrogen bonds of saturated fatty acids.Y. lo