Mori M, Vigh S, Miyata A, Yoshihara T, Oka S, Arimura A
U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, Louisiana 70037.
Endocrinology. 1990 Feb;126(2):1009-13. doi: 10.1210/endo-126-2-1009.
Although the posterior pituitary is known to contain the PRL releasing activity or factor (PRF), its chemical identification has been a matter of dispute. In the present study, we purified PRF in porcine posterior pituitary extracts to chemically determine the primary structure. PRF activity was assessed during purification by the release of immunoreactive PRL from superfused rat pituitary cells. Two hundred seventy porcine posterior pituitaries were boiled, homogenized, and extracted with 2 M acetic acid. The acid extract was precipitated with 67% acetone, and the supernatant was absorbed onto a C18 column. The column was eluted step-wise with 10, 20, 30, 40, 50, and 60% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The greatest PRF activity was recovered in the 30% CH3CN/0.1% TFA fraction and was further purified by ion-exchange chromatography on SP-Sephadex, followed by gel-filtration on Sephadex G-50. The Sephadex G-50 fractions with major PRF activity were finally purified by two cycles of reverse phase HPLC, yielding a single peak of PRF. Amino acid, as well as sequence analyses, indicated that the highly purified PRF was oxytocin. Authentic oxytocin showed the same chromatographic behavior and biological activity as those of the isolated peptide. In another experiment, desalted crude extracts of rat and porcine posterior pituitary tissues were directly chromatographed by reverse phase HPLC, and each fraction was assayed for PRF activity. Only two areas showed PRF activity; the largest activity coeluted with oxytocin and the smaller one co-eluted with vasopressin. The fractions which coeluted with oxytocin also showed oxytocin immunoreactivity, as examined by RIA. The results clearly indicated that the major PRF in these posterior pituitary extracts was oxytocin.
虽然已知垂体后叶含有催乳素释放活性或因子（PRF），但其化学鉴定一直存在争议。在本研究中，我们对猪垂体后叶提取物中的PRF进行了纯化，以化学方式确定其一级结构。在纯化过程中，通过从经超灌流的大鼠垂体细胞中释放免疫反应性催乳素来评估PRF活性。将270个猪垂体后叶煮沸、匀浆，并用2M乙酸提取。酸提取物用67%丙酮沉淀，上清液吸附到C18柱上。用0.1%三氟乙酸（TFA）中的10%、20%、30%、40%、50%和60%乙腈（CH3CN）对柱进行分步洗脱。最大的PRF活性在30%CH3CN/0.1%TFA组分中回收，并通过在SP-葡聚糖凝胶上进行离子交换色谱进一步纯化，随后在葡聚糖凝胶G-50上进行凝胶过滤。具有主要PRF活性的葡聚糖凝胶G-50组分最终通过两轮反相高效液相色谱纯化，得到单一的PRF峰。氨基酸分析以及序列分析表明，高度纯化的PRF是催产素。纯催产素显示出与分离肽相同的色谱行为和生物活性。在另一项实验中，大鼠和猪垂体后叶组织的脱盐粗提取物直接通过反相高效液相色谱进行色谱分析，每个组分都检测PRF活性。只有两个区域显示出PRF活性；最大活性与催产素共洗脱，较小的一个与加压素共洗脱。通过放射免疫分析检测，与催产素共洗脱的组分也显示出催产素免疫反应性。结果清楚地表明，这些垂体后叶提取物中的主要PRF是催产素。
