Epidermal growth factor (EGF-beta) and transforming growth factor (TGF-alpha) are structurally related and exhibit similar activities in competition for binding to the EGF receptor, stimulation of DNA synthesis, and cell proliferation. The objective of the project is to study the mechanism of their mitogenic activities based on their molecular conformations. The conformation of naturally occurring and synthetic analogues of EGF and TGF-alpha will be studied by nuclear magnetic resonance (NMR), circular dichroism (CD), laser Raman, and infrared spectroscopic methods. The radioreceptor assay and the mitogenic assay will be used for testing biological activities of the synthetic analogues. At present, the research is focused on the following areas: (1) cyclization of synthetic peptides through disulfide linkage by high dilution method, (2) purification of cyclic peptides by gel chromatography and reversed phase HPLC systems, (3) examination of the biological activities of these synthetic peptides, (4) conformational studies of synthetic analogues of EGF and TGF-alpha by modern two-dimensional NMR techniques. Results obtained so far include: (1) Isolation and purification of human EGF from urine has been achieved by ethanol precipitation,ion-exchange chromatography, gel filtration, and FPLC. Fractions active in stimulating DNA synthesis in primary rat hepatocytes were obtained. The radioreceptor assay also was used to confirm biological activities of the active fraction. However, a minute quantity of human EGF was isolated from urine. (2) Two cyclic peptides, analogues of human TGF-alpha, c(C-R-F-L-V-Q-E-D-K-P-A-C) and c(C-F-H-G-T-A-R-F-L-V-Q-E-D-K-P-A-C), have been successfully synthesized by high dilution method. The structures of cyclic peptides were confirmed by FAB mass spectrometry. (3) Conformational analysis of 17-mer is now underway.
