Separation of prolactin releasing factor from bovine posterior pituitary

牛垂体后叶提取物可诱导大鼠产生明显的催乳素释放活性，而VIP、PHI、TRH、血管紧张素II、P物质、催产素和甘丙肽等公认的大鼠催乳素释放因子（prolactin releasing factors，PRF）在牛垂体前叶组织中仅具有微弱的PRF活性。在屠宰场收集了270头牛的后垂体，用2 M乙酸提取。酸性提取物用75%丙酮沉淀，上清液经Dowex离子交换层析，依次用水、0.2M吡啶、2 M吡啶和2 M氨水洗脱。然后，将2 M吡啶级分吸收在Biogel P-4柱中用于凝胶过滤。主要的PRF活动的馏分，最后半纯化。牛垂体后叶经煮沸后，用2 M乙酸提取。用0.5 M乙酸复溶浸提液，并向浸提液中加入冷丙酮至最终浓度为75%。搅拌过夜后，通过离心沉淀。将上清液蒸发，上样于Bio-gel P-4柱上，并用0.5M乙酸洗脱。收集120个级分，并通过280 nm处的UV吸光度监测肽的洗脱曲线。以分散的垂体前叶细胞释放PRL的能力评价各组分中的PRF活性。放免法测定培养液中PRL浓度。采用凝胶过滤色谱法对垂体后叶提取液和分子量标记物进行分析，以测定PRF的分子量。从三种标记物的洗脱位置和分子量获得线性回归。PRF的分子量约为730。

Bovine posterior pituitary extracts induced a remarkable prolactin releasing activity, while VIP, PHI, TRH, Angiotensin II, Substance P, oxytocin and galanin, which are recognized as putative prolactin releasing factors (PRF) in rats, had only weak PRF activities in perifused bovine anterior pituitary explants. Two hundred seventy bovine posterior pituitaries were collected at a slaughter house, extracted with 2 M acetic acid. The acid extract was precipitated with 75% acetone, and supernatant was applied ion-exchange chromatography of Dowex and eluted stepwise water, 0.2 M pyridine, 2 M pyridine and 2 M ammonia. Then, 2 M pyridine fraction was absorbed in Biogel P-4 column for gel-filtration. The major PRF activities of the fractions were finally semipurified. Molecular weight of these fractions were less than LH-RH.Bovine posterior pituitaries were boiled and extracted with 2 M acetic acid. The extract was reconstituted with 0.5 M acetic acid, and cold acetone was added to the extract to a final concentration of 75%. After overnight stirring, the precipitate was pelleted by centrifugation. The supernatant was evaporated, applied on Bio-gel P-4 column, and eluted with 0.5 M acetic acid. One hundred twenty fractions were collected, and elution profile of peptides was monitored by UV absorbance at 280 nm. PRF activity in each fraction was evaluated with capacity of PRL release from dispersed anterior pituitary cells. PRL concentration in incubated media were measured by RIA. To estimate the molecular weight of PRF, posterior pituitary extract and molecular weight markers were carried out the gel filtration chromatography. The linear regression was obtained from the elution position and molecular weight of three markers. The molecular weight of the PRF was approximately 730.