Proteolytic processing of proopiomelanocortin (POMC) is required to produce active endogenous beta-endorphin-related opioid peptides that stimulate analgesia. The main goal is to assess the 70 kDa, PC1/3, and PC2 enzymes as candidate POMC processing enzymes by comparing POMC cleavage products, kinetics, enzyme colocalization with beta-endorphin, and effects of antisense expression on POMC processing in pituitary cells. The second goal is to assess whether POMC and proenkephalin (PE) are processed by different enzymes in antisense experiments. These in vitro and cellular studies of POMC processing are feasible because of our recent purification of endogenous POMC cleaving enzymes -- the 70 kDa aspartic proteinase, PC1/3, and PC2. Furthermore, functional rat anterior and intermediate pituitary cells in primary cultures allows examination of the role of each enzyme (in antisense studies) in cells from in vivo tissue that naturally express POMC. Firstly, in vitro processing of POMC will be compared for the 70 kDa aspartic proteinase, PC 1/3, and PC2 with respect to kinetics, cleavage sites, and beta-endorphin/ACTH products. Purified enzymes will be obtained from chromaffin granules of bovine adrenal medulla which are highly enriched in these proteases, and recombinant POMC will be expressed and purified from E. coli. POMC cleavage products will be identified by peptide microsequencing. Secondly, the secretory vesicle localization and neuroendocrine tissue distribution of these enzymes will be compared with that of beta-endorphin and ACTH by immunoelectron microscopy, immunofluorescence histochemistry, and radioimmunoassay. Thirdly, molecular cloning will isolate the 70 kDa aspartic proteinase cDNA by RT- PCR and RACE approaches based on peptide microsequencing of the enzyme; cDNA libraries will be screened with PCR generated probes, complementary oligonucleotides, and anti-aspartic proteinase antibodies. Expression in E. coli or mammalian cells (BSC40) will confirm the cDNA as the 70 kDa aspartic proteinase. Rat and mouse cDNAs will also be isolated for antisense studies in pituitary cells from these species. Lastly, the effects of enzyme antisense expression on POMC processing in anterior and intermediate pituitary cells will be compared. Results may implicate particular proteases in tissue-specific processing of POMC. Comparison of enzyme antisense studies in POMC processing cells with proenkephalin processing in chromaffin cells may indicate whether POMC and PE are processed by different proteases. These studies will provide a comprehensive evaluation of primary POMC processing enzyme(s).
| Yasothornsrikul, S; Hook, V Y (2000) Detection of proteolytic activity by fluorescent zymogram in-gel assays. Biotechniques 28:1166-8, 1170, 1172-3 |
