Isolation of Growth Hormone-Releasing Hormone (GRH)¹from Porcine Hypothalami

Abstract

Isolation is described on a preparative scale of growth hormone-releasing hormone (GRH) from 200,000 porcine hypothalami. Boiled, lyophilized 2N acetic acid extracts of hypothalamic tissue were re-extracted with glacial acetic acid. The GRH activity in the concentrate, as followed by in vivo and in vitro bioassays, was subjected to purification by several successive methods. Preparative gel filtration on a large column of Sephadex G-25 in batches of 45–70 g, followed by rechromatography in the same system, led to separation of GRH from other hypothalamic releasing hormones and from vasopressin. Subsequent purification steps included free-flow electrophoresis for 96 hr at pH 6.3, ion-exchange chromatography on carboxymethyl, diethylaminoethyl and triethylaminoethyl celluloses utilizing volatile ammonium acetate buffers, and finally partition chromatography using Sephadex as a support. The peptide with GRH activity, thus purified about 56,000 times, released GH in rats in vivo at doses of 1 nanogram and in ...