A Bovine Hypothalamic Peptide Possessing Immunoreactive Growth Hormone-Releasing Activity*

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Abstract
A systematic search for a GH-releasing factor (GHRF) was carried out using bovine hypothalamic extracts. Purification by Sephadex G-25 column chromatography yielded 17 crude polypeptide peaks distributed over 720 fractions. These were separately pooled, lyophilized, and tested in vivo in rats for GH-releasing activity. Only two peaks (X and X1) were active (50 μg) in releasing immunoreactive GH from the rat anterior pituitary. Small portions of this crude material were purified by affinity chromatography on a column packed with agarose-bound somatostatin antibody, and the resultant somatostatin-free product was active (100 ng) in rats in vivo, showing a 9-fold increase of immunoreactive GH. Thin layer chromatographic mobility of the GH-releasing peptide was different from that of a previously reported GHRF and other known peptide hormones. Lack of inactivation of the GHRF by aminopeptidase, the negative results of Edman-dansyl degradation, and the destruction of the biological activity by pyrrolidone carboxylyl peptidase indicated a pyroglutamyl N-terminus in the hormone molecule. However, carboxypeptidase inactivated the peptide, suggesting that the C-terminus is free. Inactivation by trypsin and chymotrypsin showed the presence of lysine and/or arginine and aromatic amino acids. Infusion of the GHRF into hypophyseal stalk portal vessels of male rats and observation of the ultrastructure of somatotrophs indicated massive emiocytotic activity at 1, 5, and 15 min postinfusion, with a maximum effect occurring at 1 min. Serum GH levels of the infused rats confirmed these findings.