The prolactin releasing activity of the pregnant rat hypothalamic extract (HE). was previously reported. In the present study, HE was boiled or enzymatically digested so as to investigate the properties of the prolactin releasing factor in HE.
HE was subsequently injected into puerperal lactating rats, and serum and pituitary prolactin levels were determined by double antibody radioimmunoassay. Boiling was done at 100 °C for 15 min, and enzymatic digestion was carried out with either trypsin or alpha-chymotrypsin at 37°C for 6 hrs in Tris-HC1 buffer pH 7. 8, and terminated by freezing with acetone-dry ice.
Boiled HE caused an increase in serum prolactin levels as much as non-treated HE 2 and 4 hrs after administration. After injection of tryptic digested HE, serum prolactin levels increased 3 times as much as control 4 hrs later, but pituitary prolactin levels decreased significantly 2 hrs later. After injection of chymotryptic digested HE, serum prolactin levels increased 6. 5 times as much as control 4 hrs later, but pituitary prolactin levels decreased 2 and 4 hrs later.
Chymotryptic digestion was followed by ultracentrifugation at 105,000×g for 30min, and the supernatant and precipitate were injected into the lactating rats. The supernatant of digested HE caused the highest increase of serum prolactin levels. On the other hand, the supernatant of digested HE made in pituitary prolactin levels decreased much more than the supernatant of non-digested HE. The prolactin releasing activity existed in the supernatant of the digested HE.
The present report suggests that the prolactin releasing factor in HE is heat stable, and seems to be a polypeptide and appears to be activated by solublization as a result of enzymatic digestion.
