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Evidence for Functional Localization of the Proenkephalin-Processing Enzyme, Prohormone Thiol Protease, to Secretory Vesicles of Chromaffin Cells¹

Department of Medicine, University of California-San Diego, La Jolla, California 92093; the Department of Biochemistry, Uniformed Services University of the Health Sciences (S.N.), Bethesda, Maryland 20814; and the Naval Medical Research Institute (Y.-H.K.), Bethesda, Maryland 20814

Address all correspondence and requests for reprints to: Dr. V. Hook, Department of Medicine, University of California-San Diego, 9500 Gilman Drive, #0822, La Jolla, California 92093-0822. E-mail: vhook{at}ucsd.edu

The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide 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 proenkephalin-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 immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.

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