Evidence for functional localization of the proenkephalin-processing enzyme, prohormone thiol protease, to secretory vesicles of chromaffin cells

The biosynthesis of enkephalin opioid neuropeptides as well as numerous pep tide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephal in-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by imm unoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures o f chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunof luorescence staining in neuritic extensions and cytoplasmic (perinuclear) r egions of chromaffin cells is consistent with localization to secretory ves icles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cel ls occurred upon KCl depolarization in a calcium-dependent manner, indicati ng the localization of PTP and (Met)enkephalin within regulated secretory v esicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with th e localization of PTP to functional, regulated secretory vesicles that cont ain (Met)enkephalin.