PEPTIDE BIOSYNTHETIC PROCESSING - DISTINGUISHING PROHORMONE CONVERTASES PC1 AND PC2

To determine whether manipulation of time, temperature and intragranul ar pH could be used to distinguish the actions of two subtilisin-relat ed endoproteases, PC1 and PC2, in peptide biosynthesis, the biosynthet ic processing of proneuropeptide Y (proNPY) and proopiomelanocortin (P OMC) was examined in pituitary cell lines. AtT-20 cells express PC1 an d POMC endogenously; stably transfected AtT-20 lines expressing NPY or PC2 were studied. GH3 cells express PC2 endogenously; NPY-expressing GH3 transfectants were investigated. PCI mediated rapid processing of NPY and POMC; PC1-dependent cleavages were relatively insensitive to 2 0 degrees C blockade (which al-rests secretory pathway transport at th e trans-Golgi network) and do not require an acidic intracellular comp artment (as in secretory granules). PC2 mediated much slower processin g of proNPY and POMC which was totally blocked at 20 degrees C and req uired an acidic intracellular compartment. Thus, kinetics, abolition o f intracellular pH gradients, and incubation at reduced temperatures c an be used to distinguish PC1 and PC2 actions in neuroendocrine cells.