The N-terminal portion of growth inhibitory factor is sufficient for biological activity

To determine its active site, growth inhibitory factor (GIF), a central nervous system-specific metallothionein-like protein, was digested with trypsin followed by Staphylococcus aureus protease V8 digestion. Of 5 peptide fragments separated from trypsin-digested GIF by reverse-phase high pressure liquid chromatography and gel filtration, only GIF1-26 or longer peptides showed growth inhibitory activity on cortical neurons in culture. A shorter peptide, GIF5-23, which was obtained by further digestion of GIF1-26 with V8 protease, also showed growth inhibitory activity. However, a synthetic peptide corresponding to GIF5-23 did not show growth inhibitory activity. Metal-free GIF1-26 prepared by acid treatment showed a similar level of growth inhibitory activity to that of metal-containing GIF1-26, indicating that metal in the peptide does not affect the activity. Treatment of metal-free GIF1-26 with beta-mercaptoethanol resulted in the loss of activity. The CD spectrum of beta-mercaptoethanol-treated metal-free GIF1-26 was different from that of nontreated metal-free GIF1-26. These results indicate that the N-terminal portion of GIF is required for growth inhibitory activity and that folding of the peptide via S-metal bonding is critical for biological activity.