Spannagel A W, Green G M, Guan D, Liddle R A, Faull K, Reeve J R
Department of Physiology, University of Texas Health Science Center, San Antonio 78284, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4415-20. doi: 10.1073/pnas.93.9.4415.
Cholecystokinin (CCK) secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition of CCK release caused by pancreatic proteases is due to proteolytic inactivation of a CCK-releasing peptide present in intestinal secretion. To purify the putative luminal CCK-releasing factor (LCRF), intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in conscious rats. From these secretions, the peptide was concentrated by ultrafiltration followed by low-pressure reverse-phase chromatography and purified by reverse-phase high-pressure liquid chromatography. Purity was confirmed by high-performance capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially purified fractions strongly stimulated both pancreatic secretion and CCK release while CCK receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for 41 residues as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV. When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in conscious rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRF-(1-6) abolished the CCK releasing activity of intestinal secretions. These studies demonstrate, to our knowledge, the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.
大鼠和人类体内的胆囊收缩素（CCK）分泌会受到肠道中胰蛋白酶和胆汁酸的抑制。据推测，胰蛋白酶对CCK释放的抑制作用是由于肠道分泌物中一种CCK释放肽的蛋白水解失活所致。为了纯化假定的肠腔CCK释放因子（LCRF），通过向清醒大鼠的空肠改良Thiry-Vella瘘管灌注来收集肠道分泌物。从这些分泌物中，通过超滤浓缩该肽，接着进行低压反相色谱，然后通过反相高压液相色谱进行纯化。通过高效毛细管电泳确认纯度。通过将各组分注入清醒大鼠的近端小肠后刺激胰腺蛋白分泌的能力来测定其CCK释放活性。部分纯化的组分强烈刺激胰腺分泌和CCK释放，而CCK受体阻断则消除了胰腺反应。氨基酸分析和质谱分析表明，纯化后的肽由70 - 75个氨基酸残基组成，分子量为8136 Da。LCRF的微序列分析得到了41个残基的氨基酸序列如下：STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV。当经十二指肠内注入时，纯化后的肽在清醒大鼠中以剂量相关的方式刺激胰腺蛋白和液体分泌，并显著提高血浆CCK水平。使用针对合成LCRF-(1-6)产生的抗血清进行免疫亲和色谱分析，消除了肠道分泌物的CCK释放活性。据我们所知，这些研究首次对一种作为肠腔内肠道激素释放调节因子的肠腔分泌肠肽进行了化学表征。
