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.
Uchida
Y
Y
Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Japan.
Ihara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001923:Brain Chemistry; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D000090023:Metallothionein 3; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D014357:Trypsin
D006131:Growth Inhibitors; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D014357:Trypsin