The focus of this grant application is to study a proteases involved in processing of neuropeptide precursors such as prodynorphin. A neuropeptide processing endoprotease designated """"""""dynorphin converting enzyme"""""""" capable of producing dynorphin B-13 has been previously purified and characterized. The enzyme exhibits a broad tissue-distribution suggesting involvement in the generation of a number of neuropeptides. In this grant application, the substrate specificity of this enzyme will be examined (Specific Aim 1). Synthetic peptide substrates will be used to compare its substrate specificity to the specificity of the recently identified prohormone convertases. A cDNA clone encoding the dynorphin converting enzyme will be isolated (Specific Aim 2). Polymerase chain reaction with oligonucleotides based on cysteine protease consensus sequences has led to the identification of four novel cDNAs; one of these shows tissue-specific expression which generally matches the distribution of the dynorphin converting enzyme. The full length cDNA will be isolated and expressed and the expressed protein will be characterized using the various substrates proposed in Specific Aim 1. The distribution of the mRNA by in situ hybridization analysis will also be examined.
