Study on the function of endogenous opioid and substance P at inflammation and pain

我们合成了Spin类似物，并测定了它们对脑啡肽降解酶中的二肽基多肽酶III(DPPIII)的抑制活性。VVYPW是一种N-端和C-端截短形式的Spin，显示出更强的抑制活性。这一结果表明VVYPW对DPPIII具有更有效的抑制活性表达结构。我们试图将伤害性感觉纤维分为三种类型，并与吗啡诱导的疼痛相比较，考察了Spin诱导的镇痛的作用方式。因此，Spin完全阻断了2-甲基硫代腺苷的反应，而吗啡没有。另一方面，经百日咳毒素处理后，吗啡对缓激肽的阻断作用减弱，而脊髓吗啡的抑制作用不明显。因此，提示Spin具有广泛的镇痛作用，包括对吗啡不敏感的伤害性感觉的阻断。针对人P物质受体的纯化，我们研制了N端带有6xHis表位的重组P物质受体。6xHis标签不干扰受体与配体的结合能力，6xHis-SPR可通过Ni-NTA柱纯化。这一纯化过程是获得大量用于结构研究的均相SPR的重要一步。

We synthesized spinorphin analogous and assayed their inhibitoriy activity toward dipeptidyl peptidase III (DPPIII) among enkephalin degrading enzymes. VVYPW, an N-terminal and C-terminal truncated form of spinorphin, exhibited more potent inhibitory activity.This results indicated that VVYPW had amore effective structure of expression of inhibitory activity toward DPPIII.We attempted to characterize nociceptive sensory fibers into three types, and examined the mode of action of spinorphin-induced analgesia, in comparison to morphine-induced one.So, spinorphin completely blocked 2-metyl-thioadenosine induced responsed, but morphine did not. On the other hand, morphine-induced blockade of bradykinin responses was attenuated by pertussis toxin treatment, while the spinorphin's ones was not. Thus it is suggested that spinorphin has wide spectrum of analgesia which covers the blockade of nociception insensitive to morphine.Regarding to purification of human substance P receptor, we have developed a recombinant Substance P receptor with 6xHis epitope at its N-terminus. The 6xHis tag does not interfere with receptor's ability to bind ligands the 6XHis-SPR can be purified over the Ni-NTA column. This purification procedure constitutes an important step toward obtaining large quantities of homogeneous SPR for structural studies.