Mitra Sankar P
Indian J Biochem Biophys. 2013 Dec;50(6):511-20.
The study focuses on the importance of Tyr11 amino acid (AA) and subsequent stereochemistry involved in the binding process of neurotensin (NT) with its receptor (NTR)/binding protein(s) as well as the size heterogeneity. Using the binding of 125I-NT with several chicken tissues, it is identified that one of the crucial factors behind all high affinity (Kd -10 pM) interactions is due to phenolic-OH (D-OH) at the para (p) position of Tyr11 within RRPYIL-CO2H (NT8-13) sequence. Replacing the p-OH only in Tyr11 by substituting with p-C1, p-F and p-NH2 results in significant change of the binding affinity (Kd); p-OH approximately equal p-NH2 (approximately 10 pM), p-Cl (approximately 100 pM), p-F (approximately 120 pM). Interestingly, p-NH2 equals to p-OH displaying the highest affinity. Experiments conducted by binding several of the 125I-azido-NT analogs having azido group attached at different positions within the NT molecule have further confirmed the necessity of RRPYIL sequence for high affinity ligand-receptor interaction. The role of Tryp11 in place of Tyr11 in addition to the results above establishes a significant possibility of H-bonding occurring between p-OH of NT and NTR inside the docking space. Photo labeling of the liver tissue by substituted 125I-Y3-azido-NT analogs shows several specifically labeled bands with considerable range of molecular weight (Mr approximately 90-30 kDa) variations. These results indicate the existence of molecular heterogeneity concerning the sizes of NTR or else any NT binding proteins in the avian tissues. Further, the study has revealed that besides liver, several other chicken tissues also express similar specific high affinity binding (Kd approximately 20 pM) with varying capacities (Bmax). The order for Bmax is: liver (1.2 pMol/mg) > or = gall bladder (1.03 pMol/mg) > spleen (0.43 pMol/mg) > brain (0.3 pMol/mg) > colon > or = lung (0.15 pMol/mg). In all cases, the binding was reduced by GTPgammaS (ED50 to approximately 0.05 nM), NEM (ED50 to approximately 0.50 mM) and NaCl (ED50 to approximately 30 mM), indicating the existence of NTR identical to the mammalian type-1.
该研究聚焦于酪氨酸11氨基酸（AA）及其后续立体化学在神经降压素（NT）与其受体（NTR）/结合蛋白结合过程中的重要性以及大小异质性。通过125I-NT与几种鸡组织的结合实验，确定所有高亲和力（Kd -10 pM）相互作用背后的关键因素之一是RRPYIL-CO2H（NT8-13）序列中酪氨酸11对位（p）的酚羟基（D-OH）。仅将酪氨酸11中的对羟基分别用对氯、对氟和对氨基取代，会导致结合亲和力（Kd）发生显著变化；对羟基约等于对氨基（约10 pM），对氯（约100 pM），对氟（约120 pM）。有趣的是，对氨基与对羟基显示出最高的亲和力。通过结合几种在NT分子内不同位置连接有叠氮基的125I-叠氮基-NT类似物进行的实验进一步证实了RRPYIL序列对于高亲和力配体-受体相互作用的必要性。除上述结果外，用色氨酸11替代酪氨酸11的作用表明在对接空间内NT的对羟基与NTR之间存在氢键形成的显著可能性。用取代的125I-Y3-叠氮基-NT类似物对肝脏组织进行光标记显示出几条特异性标记带，分子量（Mr约90 - 30 kDa）有相当大的变化范围。这些结果表明禽类组织中NTR或其他任何NT结合蛋白在大小方面存在分子异质性。此外，该研究还揭示，除肝脏外，其他几种鸡组织也表达类似的特异性高亲和力结合（Kd约20 pM），但结合能力（Bmax）各不相同。Bmax的顺序为：肝脏（1.2 pMol/mg）≥胆囊（1.03 pMol/mg）＞脾脏（0.43 pMol/mg）＞脑（0.3 pMol/mg）＞结肠≥肺（0.15 pMol/mg）。在所有情况下，GTPγS（ED50约为0.05 nM）、NEM（ED50约为0.50 mM）和NaCl（ED50约为30 mM）都会降低结合，表明存在与哺乳动物1型相同的NTR。
