Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.
Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.
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鉴定磷脂酰胆碱转移蛋白与含有光活化卡宾前体的磷脂酰胆碱类似物的脂质结合位点。
DOI：
10.1111/j.1432-1033.1983.tb07382.x
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Khorana,HG
中科院分区：
文献类型：
--
作者：
Westerman,J;Wirtz,KW;Berkhout,T;vanDeenen,LL;Radhakrishnan,R;Khorana,HG
用带有二氮杂环苯氧基的磷脂酰胆碱类似物研究了牛肝脏磷脂酰胆碱转运蛋白的脂结合部位。该二氮杂环苯氧基连接在n-2-[1-14C]己酰(PCI)或n-2-[1-14C]十一碳(PC II)的ω碳上。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法显示，PCII-转移蛋白复合体的光解导致30-40%的标记与蛋白质发生共价偶联。对光解的络合物进行弱碱处理后，用凝胶渗透色谱将含有共价偶联的14C标记的蛋白质从非偶联的14C标记中分离出来。用来自金黄色葡萄球菌、胰酶和溴化氰的酶降解14C标记的蛋白质，并用自动Edman降解法对14C标记的多肽进行测序。放射性释放显示主要偶联部位为Tyr54和肽段Val171-Phe-Met-Tyr-Tyr-Phe-Asp177。PC I和PC II都广泛地与Tyr54偶联(分别占总标记的90%和5%)。其余的放射性是从Val171-Asp177肽中释放出来的，释放模式明显不同，这取决于使用的是PC I还是PC II。因此，Val171和Met173优先与PC I偶联，而Val171和Met173优先与PC II偶联。假设Val171-Asp177肽采用了强预测的β-链构型，这种偶联转变与如果OC I和II时n-2-脂肪-acyk cgub增加0.6 nm是相容的。这些数据被解释为磷脂酰胆碱在磷脂酰胆碱转移蛋白中的定位。
The lipid binding site of the phosphatidylcholine transfer protein from bovine liver has been investigated by use of phosphatidylcholine analogs which carry a diazirinophenoxy group linked to the ω‐carbon of either thesn‐2‐[1‐14C]hexanoyl (PCI) orsn‐2‐[1‐14C]undecanoyl chain (PC II). Photolysis of the PCI(PCII)‐transfer protein complex resulted in a covalent coupling of 30–40% of the label to the protein as shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Upon mild alkaline treatment of the photolysed complex the protein containing covalently coupled14C‐label was separated fro the noncoupled14C‐label by gel permeation chromatography. The14C‐labeled protein was degraded with protease fromStaphylococcus aureus, trypsin and cyanogen bromide and specific14C‐labeled peptides were sequenced by automated Edman degradation. Major sites of coupling shown by release of radioactivity were identified as Tyr54and the peptide segment Val171‐Phe‐Met‐Tyr‐Tyr‐Phe‐Asp177. Both PC I and PC II coupled extensively to Tyr54(90% and 5% of total labeling, respectively). The remainder of the radioactivity was released from the peptide Val171‐Asp177with a distinct difference in in the pattern of release depending on whether PC I or PC II were used. Thus, coupling occurred preferentially to Tyr175and Asp177with PC I while Val171and Met173were labeled preferentially with PC II. This shift in coupling is compatible with an increasae of 0.6 nm for thesn‐2‐fatty‐acyk cgaubs if OC I and II, assuming that the peptide Val171‐Asp177has adopted the strongly predicted β‐strand configuration. These data have beeninterpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.