α1‐Antichymotrypsin‐Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme “Prohormone Thiol Protease”

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Abstract
Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary α1‐antichymotrypsin (ACT), referred to as ACT‐like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56‐ and 60‐kDa ACT‐like proteins I and II, respectively, to enkephalin‐containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE‐Sepharose and chromatofocusing resulted in the separation of ACT‐like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56‐kDa ACT‐like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60‐kDa ACT‐like protein II was isolated by butyl‐Sepharose, Sephacryl S200, and concanavalin A‐Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT‐like protein I, with a Ki,app of 35 nM, but ACT‐like protein II was less effective. ACT‐like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT‐like proteins that differentially inhibit PTP. The colocalization of the ACT‐like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT‐like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.