Evidence for the proenkephalin processing enzyme prohormone thiol protease(PTP) as a multicatalytic cysteine protease complex: Activation by glutathione localized to secretory vesicles

The cysteine protease known as "prohormone thiol protease" (PTP) has been i dentified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease c omplex that can be activated by endogenous glutathione present in chromaffi n granules. The high molecular mass nature of PTP, of approximately 185 kDa , was demonstrated by elution of a single peak of S-35-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of S-35-enkephalin precursor cleaving activity detected on rad iozymogram gels under native buffer conditions. importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three ba nds of proteolytic activity with apparent molecular masses of 88, 81, and 6 1 kDa. These activities were all cysteine proteases, since they were inhibi ted by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purifica tion of native PTP by preparative gel electrophoresis indicated that PTP wa s composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-P AGE gels. These four protein subunits accounted for the three catalytic act ivities of PTP, as demonstrated on S-35-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the fou r subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing condition s for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin gran ules that may be involved in the proteolytic processing of proenkephalin an d perhaps other precursors into active neuropeptides.