| Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.;GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G;In the work reported here, hundred mg quantities of pro-GnRH/GAP were prepared by novel methods of both chemical synthesis and bacterial expression.;An immunoassay was developed against a processing site epitope within pro-GnRH/GAP. Both synthetic and recombinant pro-GnRH/GAP proteins are immunoreactive, consistent with the idea that the epitope, and, thus, the processing site, is located on the surface of the molecule. Proteolysis of synthetic or recombinant pro-GnRH/GAP by trypsin or kallikrein caused immediate loss of immunoactivity, showing that the processing site is susceptible to proteolysis and that the integrity of the processing site is required for immunoactivity. One of the kallikrein hydrolytic products was identified as GAP. Therefore, kallikrein cleaves at the R;The intrinsic fluorescence yield of the Trp residue near the processing site region of pro-GnRH/GAP is sensitive to changes in pH, but not to changes in ionic strength or calcium concentration; its fluorescence yield is maximal at neutral pH.;Thermal denaturation of pro-GnRH/GAP follows a simple two-state transition at neutral pH, as assessed by differential scanning calorimetry. (Abstract shortened by UMI.). |
