Separation of prolactin releasing factor from bovine posterior pituitary

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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.