Proteolytic processing of inactive proenkephalin is required to produce active endogenous opioid enkephalin peptides. the goal of this proposal is to examine the relative roles of several proenkephalin (PE) processing enzymes -- (a) a novel 'prohormone thiol protease', (b) PC2 and PC3 prohormone convertases, and (c) carboxypeptidase H -- to determine which of these is the primary and rate-limiting enzyme in enkephalin biosynthesis. We have identified a novel 'prohormone thiol protease', PTP, that is involved in PE processing. We also demonstrate PC2 and PC3 (PC = prohormone convertase) activities in enkephalin-containing chromaffin granules, implicating a role for these subtilisin-like proteases in PE processing. with these findings, specific aims 1 and 2 will compare PTP, PC2, and PC3 activities with respect to the order to PE cleavages and products generated, as well as determine enzyme kinetic constants to assess substrate affinity and maximal enzyme activity. These in vitro studies of PE processing enzymes will utilize as substrate recombinant PE generated by high level expression in E. Coli. Expression of the PE cDNA provides milligram amounts of PE that allows definitive identification of PE products by peptide microsequencing and amino acid compositional analyses. Importantly, adequate levels of recombinant PE also allows analysis of the kinetic constants, Km and Vmax, near in vivo levels of PE. Subsequent to endoproteolytic processing of PE, carboxypeptidase H is required to remove COOH-terminal basic residues. The processing of the CPH precursor, pro-CPH, like other proteolytic cascades (such as in blood clotting) may be catalyzed by a protease involved in one of the first steps of processing. Therefore, specific aim 3 will assess processing of pro-CPH by purified PTP, PC2, and PC3. To obtain a more complete understanding of CPH biosynthesis, specific aim 3 will also characterize multiple CPH mRNAs by RT-PCR (reverse transcriptase-polymerase chain reaction), DNA sequence analysis of CPH cDNAs, and limited DNA sequence analysis of genomic clones to assess if CPH transcripts arise from differential transcriptional initiation sites, alternative RNA splicing, or choice of polyadenylation site(s). These studies will provide a comprehensive study of the roles of PTP, PC2, PC3, and CPH in proenkephalin processing, and will indicate the rate-limiting PE processing enzyme whose regulation should be investigated in future investigations. These studies may lead to identification of target enzyme(s) for development of therapeutic drugs that modify the opiate system through PE processing.
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