Binding of substance P agonists to lipid membranes and to the neurokinin-1 receptor.
Binding of substance P agonists to lipid membranes and to the neurokinin-1 receptor.
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P 物质激动剂与脂膜和神经激肽-1 受体的结合。
DOI：
10.1021/bi952434q
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
G. Hölzemann
中科院分区：
文献类型：
--
作者：
A. Seelig;T. Alt;S. Lotz;G. Hölzemann
合成了神经肽P物质(SP)的三个新类似物。C末端信息片段在(Arg9)SP中更亲水，在(Nle9)SP中更疏水。在(AcPro2，Arg9)SP中，N端地址段的费用降低，而消息段的费用增加。这些取代的基本原理是将SP类似物的物理化学性质，特别是它们的脂质诱导构象和膜结合亲和力与受体结合和功能活性联系起来。圆二色谱表明，在溶液中，三个类似物均呈无规卷曲构象。十二烷基硫酸钠胶束的加入诱导了部分α螺旋结构的形成。(AcPro2，Arg9)SP和(Arg9)SP也由带负电荷的脂泡形成相同的结构，而SP和(Nle9)SP既有α-螺旋结构，又有β-折叠结构。吉布斯吸附等温线的测量和单层膨胀研究提供了关于SP类似物的表面积需求和膜渗透面积的定量数据。用单层膨胀法和高灵敏滴定量热法测定了脂质结合的热力学参数。含100%POPG的膜的表观结合常数Kapp为10(3)-10(5)M(-1)量级。这种结合是由于阳离子多肽对带负电的膜表面的静电吸引。(Arg9)SP的Kp=10+/-1M(-1)，(AcPro2，Arg9)SP的Kp=9+/-1M(-1)，(Nle9)SP的Kp=39+/-3M(-1)。与NK-1受体结合的亲和力和体外活性的测定表明，这三种多肽都具有激动剂的作用。它们与神经激肽-1受体的结合亲和力随着氨基酸序列第9位侧链的大小而降低，但与多肽的阳离子电荷无关。事实上，即使是高电荷(Arg9)的SP也具有激动剂活性，这一事实提供了证据，表明受体的结合表位处于相当亲水的环境中。这一发现与这三种多肽的低疏水结合常数和对负电荷膜的微弱渗透是一致的。它反对膜介导的受体机制，并认为激动剂从水相接近受体结合部位。
Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase.