title: Stimulation of "prohormone thiol protease" (PTP) and [Met]enkephalin by forskolin. Blockade of elevated [Met]enkephalin by a cysteine protease inhibitor of PTP.
abstract: Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.
authors.affiliation: Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
authors.forename: N
authors.initials: N
authors.lastname: Tezapsidis
authors.forename: S
authors.initials: S
authors.lastname: Noctor
authors.forename: R
authors.initials: R
authors.lastname: Kannan
authors.forename: T J
authors.initials: TJ
authors.lastname: Krieger
authors.forename: L
authors.initials: L
authors.lastname: Mende-Mueller
authors.forename: V Y
authors.initials: VY
authors.lastname: Hook
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000187:drug effects / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004744:Enkephalin, Methionine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D015853:Cysteine Proteinase Inhibitors; D011498:Protein Precursors; D005576:Colforsin; D004744:Enkephalin, Methionine; D000242:Cyclic AMP; D003546:Cysteine Endopeptidases; C083275:prohormone thiol protease; D007930:Leucine; C108192:aloxistatin