Spiess J, Rivier J, Rivier C, Vale W
Proc Natl Acad Sci U S A. 1981 Oct;78(10):6517-21. doi: 10.1073/pnas.78.10.6517.
Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.
对一种绵羊下丘脑41个氨基酸残基的多肽进行了序列分析。由于其具有较高的内在促肾上腺皮质激素释放活性，该多肽被推测为一种假定的促肾上腺皮质激素释放因子（CRF）。CRF的NH2末端39个残基通过在Wittmann-Liebold改良的贝克曼890C旋转杯测序仪中对0.6 - 3.5 nmol的肽进行埃德曼降解，并结合反相高压液相色谱法来鉴定氨基酸苯硫乙内酰脲（直接微量序列分析）来确定。通过在分析前用胰蛋白酶选择性地在其精氨酸残基处切割2.0 nmol的乙酰化CRF进行直接微量序列分析，提供了第40位残基（异亮氨酸）的证据。热解COOH末端片段异亮氨酰 - 丙氨酰胺被表征为其丹磺酰衍生物。基于分析数据，提出了绵羊下丘脑CRF的以下一级结构：H - Ser - Gln - Glu - Pro - Pro - Ile - Ser - Leu - Asp - Leu - Thr - Phe - His - Leu - Leu - Arg - Glu - Val - Leu - Glu - Met - Thr - Lys - Ala - Asp - Gln - Leu - Ala - Gln - Gln - Ala - His - Ser - Asn - Arg - Lys - Leu - Leu - Asp - Ile - Ala - NH2。与该提议一致，CRF的合成复制品在刺激促肾上腺皮质激素和β - 内啡肽样免疫活性的分泌方面具有高效力。
