In collaboration we have developed a series of small synthetic growth hormone releasing peptides by a closely integrated process of theoretical conformational calculations, synthesis of model peptides and assessment of biological activity. One of the most important of these peptides was the synthetic hexapeptide GH-RP, His-DTrp-Ala-Trp-DPhe-Lys-NH2, because of its high potency, specificity of action and unique actions on the pituitary (P) and the hypothalamus (H). In vitro and in vivo evidence supports that GH-RP mediates GH release via non-GRF, non-opiate receptors. Since it has all the functional and molecular attributes of a hypothalamic hypophysiotrophic hormone, it is our hypothesis that GH-RP reflects the activity of a native hormone which is present in the H and thus, it is the objective of this proposal to isolate, elucidate the structure, and synthesize the putative native peptide. Key to the strategies, approaches and techniques which will be utilized to isolate the putative native GH-RP is the in-depth knowledge we have accumulated about the structure activity relationships of synthetic GH-RP peptides. Besides utilization of conventional chromatography techniques and schemes well established for the isolation of hypothalamic hormones along with established highly sensitive in vitro and in vivo bioassays to detect the activity of the peptide, a detailed, in-depth immunological scheme has been proposed which should substantially facilitate the isolation. Monoclonal and polyclonal antibodies will be generated to the conformation of GH-RP. These conformational antibodies will allow the development of a GH-RP RIA and RRA which can be utilized for immunodetection of the putative peptide during its isolation as well as reagents for immunopurification by affinity chromatography, immunoneutralization activity during H+P incubations and immunoprecipitation of biosynthetically labelled native GH-RP during H+P incubations. Not only will these latter studies help to further establish the existence of native GH-RP but it is envisioned that select chemical/functional characteristics will be revealed on this microscale that will be of specific value in guiding the isolation of native GH-RP on the more conventional macroscale isolation schemes proposed. This work perhaps will be representative of another approach on how new endogenous peptide hormones might be revealed.
