Further Purification of TSH-Releasing Factor (TRF) from Sheep Hypothalamic Tissues,with Observations on the Amino Acid Composition. ∗

Summary

Eighty thousand hypothalamus fragments of sheep brains (wet weight 55 kg) were extracted with 2 N acetic acid. The extract was filtered on Sephadex G25 in pyridine acetate 0.1 M, pH 5.6. The TRF-active zone was located by bioassay and further purified by CCD (250 transfers followed by 1,000 transfers) using the system n-butanol-pyridine-acetic acid (5:3:11), ion exchange on IRC-50 equilibrated with ammonium acetate 0.02 M, pH 5.0, and finally thin-layer chromatography on cellulose (butanol-acetic acid-water, 4:1:5). The material obtained (ca 400 μg) at the last stage of purification was active in vivo to stimulate release of TSH at ≤ 0.1 μg dry weight. Highly purified TRF has no LH- or ACTH-releasing activity, Amino acids present in this preparation are reported.