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胰腺多肽(Pancreatic\Pancreastatin)定义

胰多肽（PP）是由十二指肠胰腺内分泌细胞分泌的一种激素。

Pancreatic polypeptide (PP) is a hormone produced by endocrine cells of the duodenal pancreas.

胰抑素（PST）是一种对分泌具有普遍抑制作用的调节肽，来源于嗜铬粒蛋白A，这是一种广泛存在于神经内分泌系统中的糖蛋白。嗜铬粒蛋白A（CgA）也被称为分泌蛋白I，是一种由许多神经内分泌细胞和神经元表达的酸性蛋白。

Pancreastatin (PST) is a regulatory peptide with a general inhibitory effect on secretion, is derived from chromogranin A, a glycoprotein present throughout the neuroendocrine system. Chromogranin A (CgA) is also referred to as secretory protein I, is an acidic protein expressed by many neuroendocrine cells and neurons.

胰腺多肽(Pancreatic\Pancreastatin)的结构特征

PP含有36个氨基酸，分子量为4.2 kDa，等电点为pH 6至7。肽1的氨基酸序列为Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2。人CgA是一种由439个残基组成的蛋白质，前面有一个由18个残基组成的信号肽。人CgA与牛CgA的蛋白质序列比较显示，NH2端和COOH端结构域以及潜在的二碱基切割位点高度保守，而中间部分则表现出显著的序列变异（36%）。人CgA中含有的PST序列两侧是蛋白水解加工位点。这表明人CgA可能是人胰抑素分子的前体，也可能是其他尚未鉴定的生物活性肽的前体【2】。

PP contains 36 amino acids, has a molecular weight of 4.2 kDa and the isoelectric point is pH 6 to 7. The amino acid sequence of the peptide 1 is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.  The human CgA is a 439-residue protein preceded by an 18-residue signal peptide. Comparison of the protein sequence of human CgA with that of bovine CgA shows high conservation of the NH2-terminal and COOH terminal domains as well as the potential dibasic cleavage sites, whereas the middle portion shows remarkable sequence variation (36%). The PST sequence contained in human CgA is flanked by sites for proteolytic processing. This suggests that human CgA may be the precursor for a human Pancreastatin molecule and possibly for other, as yet unidentified, biologically active peptides 【2】.

胰腺多肽(Pancreatic\Pancreastatin)的作用机制

PP通过其特异性受体分泌并控制分化：在一项研究中，使用小鼠转化成骨细胞系MC3T3-E1细胞研究了PP及其Y受体的调节作用。研究发现，在MC3T3-E1细胞中，细胞接触诱导分化过程中可检测到PP mRNA并增加。此外，在分化过程中，所有类型的NPY家族受体mRNA（Y1、Y2、Y4、Y5和Y6）均增加。还发现，PP在碱性磷酸酶（ALP）mRNA和骨形态发生蛋白-2（BMP-2）mRNA方面刺激了MC3T3-E1细胞的分化。这些发现表明，MC3T3-E1细胞产生并分泌PP，而PP可能通过其特异性受体以自分泌方式刺激MC3T3-E1的分化[3]。PST是一种CgA衍生的肽，已被发现可调节大鼠脂肪细胞中的葡萄糖、脂质和蛋白质代谢。PST通过激活特定的受体-效应子系统（Gaq/11蛋白-PLC-β-PKC经典途径）对胰岛素作用产生总体反调节作用。然而，PST可刺激大鼠脂肪细胞中的基础和胰岛素介导的蛋白质合成。PST剂量依赖性地刺激S6激酶的Thr421/Ser424磷酸化。此外，PST还促进4E-BP1（PHAS-I）中调节位点的磷酸化（Thr37、Thr46）。起始因子eIF4E本身在磷酸化后活性也会增加，在PST刺激下，其Ser209位点被磷酸化。此外，已有研究表明，通过阻止PKC的激活，可以阻断PST对S6激酶和翻译机制的影响。这些结果表明，PST通过激活PKC来刺激蛋白质合成机制，并为所涉及的分子机制提供了一些证据，即S6K的激活和4E-BP1（PHAS-I）以及起始因子eIF4E的磷酸化【4】。

PP is secreted from and controls differentiation through its specific receptors: In a study, the regulatory roles of PP and its Y receptors were studied using MC3T3-E1 cells, a murine transformed osteoblastic cell line. It was found that PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells. Furthermore, all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation. Also it was found that PP stimulated differentiation in MC3T3-E1 cells in terms of alkaline phosphatase (ALP) mRNA and bone morphogenetic protein-2 (BMP-2) mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner 3. PST, a CgA-derived peptide, has been found to modulate glucose, lipid, and protein metabolism in rat adipocytes. PST has an overall counter regulatory effect on insulin action by activating a specific receptor–effector system (Gaq/11protein-PLC-ß-PKC classical). However, PST stimulates both basal and insulin-mediated protein synthesis in rat adipocytes. PST dose-dependently stimulates Thr421/Ser424 phosphorylation of S6 kinase. Moreover, PST promotes phosphorylation of regulatory sites in 4E-BP1 (PHAS-I) (Thr37, Thr46). The initiation factor eIF4E itself, whose activity is also increased upon phosphorylation, is phosphorylated in Ser209 by PST stimulation. Also, it has been shown that  that these effects of PST on S6 kinase and the translation machinery can be blocked by preventing the activation of PKC. These results indicate that PST stimulates protein synthesis machinery by activating PKC and provides some evidence of the molecular mechanisms involved, i.e., the activation of S6K and the phosphorylation of 4E-BP1 (PHAS-I) and the initiation factor eIF4E 【4】.

胰腺多肽(Pancreatic\Pancreastatin)的功能

小鼠胰多肽调节食物摄入：本研究旨在探讨合成小鼠胰多肽（mPP）对小鼠进食和焦虑行为的影响。研究发现，侧脑室（i.c.v.）注射mPP（0.003-3 nmol）可剂量依赖性地增加食物摄入量。注射后20分钟即可观察到显著增加，并持续4小时。此外，腹腔（i.p.）注射mPP（0.03-30 nmol）可剂量依赖性地减少食物摄入量。注射后20分钟即可观察到显著减少，并持续4小时。在高架十字迷宫测试中，侧脑室注射mPP（0.003-3 nmol）对焦虑行为无影响。这些结果表明，mPP可调节食物摄入量，且脑内的Y4受体可能参与进食行为的调节，但似乎不影响小鼠的焦虑行为【5】。

Mouse pancreatic polypeptide modulates food intake: A study was conducted to investigate the effects of synthetic mouse pancreatic polypeptide (mPP) on feeding and anxiety in mice. It was found that the intracerebroventricular (i.c.v.) injection of mPP (0.003-3 nmol) dose-dependently increased food intake. A significant increase was observed 20 min after i.c.v. injection and continued for 4 h. Further the intraperitoneal (i.p.) injection of mPP (0.03-30 nmol) dose-dependently decreased food intake. A significant decrease was observed 20 min after i.p. injection and continued for 4 h. In the elevated plus maze test, the i.c.v. injection of mPP (0.003-3 nmol) did not affect anxiety behavior. These results suggest that mPP modulates food intake and the Y4 receptor in the brain may contribute to the regulation of feeding, whereas appearing not to influence anxiety in mice 【5】.

PST和CgA对不同促胰岛素剂刺激的胰岛素释放的影响：使用离体灌流大鼠胰腺，比较了猪胰高血糖素样肽（PST）对促胰岛素剂、胰高血糖素、八肽胆囊收缩素（CCK-8）、胃抑肽素（GIP）和L-精氨酸刺激的胰岛素释放的影响，以及牛嗜铬粒蛋白A（CGA）的影响。PST显著增强了胰高血糖素刺激的胰岛素释放（对照组第一阶段：12.5±0.9 ng/8分钟；第二阶段：34.5±1.6 ng/25分钟；胰抑素组第一阶段：16.5±1.1 ng/8分钟；第二阶段：44.0±2.2 ng/25分钟），而CGA则无效。同样，CGA也不影响CCK-8或GIP刺激的胰岛素释放。这些发现表明，PST在胰高血糖素存在时刺激胰岛素释放。由于PST对胰岛素释放有多种影响，且这些影响取决于胰岛素效应剂的局部浓度，因此PST可能参与B细胞胰岛素释放的精细调节【6】。

Effects of PST and CgA on insulin release stimulated by various insulinotropic agents: The effects of porcine PST on insulin release stimulated by insulinotropic agents, glucagon, cholecystokinin-octapeptide (CCK-8), gastric inhibitory polypeptide (GIP) and L-arginine, were compared to those of bovine chromogranin A (CGA) using the isolated perfused rat pancreas. PST significantly potentiated glucagon-stimulated insulin release (first phase: 12.5 ± 0.9 ng/8 min; second phase: 34.5 ± 1.6 ng/25 min in controls; 16.5 ± 1.1 ng/8 min and 44.0 ± 2.2 ng/25 min in pancreastatin group), whereas CgA was ineffective. Similarly, CGA did not affect insulin release stimulated by CCK-8 or GIP. These findings suggest that PST stimulates insulin release in the presence of glucagon. Because PST can have multiple effects on insulin release, which are dependent upon the local concentration of insulin effectors, PST may participate in the fine tuning of insulin release from B cells 【6】

胰腺多肽(Pancreatic\Pancreastatin)的相关文献

1.     Kimmel JR, Hayden LJ, Pollock HG (1975). Isolation and characterization of a new pancreatic polypeptide hormone. J. Biol. Chem., 250(24):9369-9376.

2.     Konecki DS, Benedum UM, Gerdes HH, Huttner WB (1987). The Primary Structure of Human Chromogranin A and Pancreastatin. J. Biol. Chem., 262(35):17026-17030.
3.     Hosaka H, Nagata A, Yoshida T, Shibata T, Nagao T, Tanaka T, Saito Y, Tatsuno I (2008). Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells. Peptides, 29(8):1390-1395.
4.     González-Yanes C, Sánchez-Margalet V (2002). Pancreastatin, a chromogranin A-derived peptide, activates protein synthesis signaling cascade in rat adipocytes. Biochemical and Biophysical Research Communications, 299(4):525-531.
5.      Asakawa A (1999). Mouse pancreatic polypeptide modulates food intake, while not influencing anxiety in mice. Peptides, 20(12):1445-1448.
6.      Ishizuka J, Tatemoto K, Cohn DV, Thompson JC, Greeley GH Jr (1991). Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents. Regulatory Peptides, 34(1):25-32.

二硫键广泛存在与蛋白结构中，对稳定蛋白结构具有非常重要的意义，二硫键一般是通过序列中的2个Cys的巯基，经氧化形成。
 

形成二硫键的方法很多：空气氧化法，DMSO氧化法，过氧化氢氧化法等。
 

二硫键的合成过程，  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
   

如果多肽中只含有1对Cys，那二硫键的形成是简单的。多肽经固相或液相合成，然后在pH8-9的溶液中进行氧化。      
 

当需要形成2对或2对以上的二硫键时，合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成，但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线：
 

二硫键反应条件选择    
 

 二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基（-SH）被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键，可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大，空间结构复杂，结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外，多肽结构中还原态的巯基化学性质活泼，容易发生其他的副反应，而且肽链上其他侧链 也可能会发生一系列修饰，因此，肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素， 反应机理也比较复杂，既可能是自由基反应，也可能是离子反应。      

反应条件有多种选择，比如空气氧化，DMSO氧化等温和的氧化过程，也可以采用H2O2，I2， 汞盐等激烈的反应条件。
 

空气氧化法： 空气氧化法形成二硫键是多肽合成中最经典的方法，通常是将巯基处于还原态的多肽溶于水中，在近中性或弱碱性条件下（PH值6.5-10），反应24小时以上。为了降低分子之间二硫键形成的可能，该方法通常需要在低浓度条件下进行。
 

碘氧化法：将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘进行氧化，反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时，氧化条件要控制的更精确，氧化完后，立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时，通常有两种情况：
 

自然随机成环:       序列中的Cys之间随机成环，与一对二硫键成环条件相似；
 

定点成环：       定点成环即序列中的Cys按照设计要求形成二硫键，反应过程相对复杂。在 固相合成多肽之前，需要提前设计几对二硫键形成的顺序和方法路线，选择不同的侧链 巯基保护基，利用其性质差异，分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。