IN-VITRO PROCESSING OF PROOPIOMELANOCORTIN BY RECOMBINANT PC1 (SPC3)

The prohormone convertases, PC1 (SPC3) and PC2, are subtilisin-like se rine proteases capable of processing neuropeptide precursors. In cotra nsfection experiments, other investigators have found that PC1 and PC2 can process POMC to appropriate peptide products. In this study, reco mbinant rat PC1 was stably expressed in a mouse L-cell line and partia lly purified. Mouse POMC was cleaved by recombinant PC1 to generate AC TH intermediates, ACTH, ACTH linked to joining peptide, joining peptid e, 16-kilodalton N-POMC, N-POMC-(1-74), and beta-lipotropin. Recombina nt PC1 was also found to cleave ACTH to ACTH-(1-15) and bovine N-POMC- (1-77) to gamma(3)MSH. The pH optimum of the cleavages was 6.0. We con clude that recombinant PC1 is capable of processing POMC in vitro at a ll of the paired basic residues, with the exception of Lys-Arg and Lys -Lys in beta-lipotropin and beta-endorphin, respectively. This in vitr o study showed a more general specificity of recombinant PC1 for paire d and tetrabasic residues of POMC than was previously found in cotrans fection experiments. Other cellular regulatory mechanisms probably pla y a role in limiting the processing of POMC in vivo in the anterior pi tuitary, where gamma(3)MSH and alpha MSH are not found in significant amounts.