alpha(1)-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"

Proteolytic processing of inactive proenkephalin and proneuropeptides is es sential for the production of biologically active enkephalins and many neur opeptides. The incomplete processing of proenkephalin in adrenal medulla su ggests that endogenous protease inhibitors may inhibit proenkephalin proces sing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha(1)-antichymotrypsin (ACT), referre d to as ACT-like proteins I and II, which are colocalized with enkephalin i n chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP), Subcellular fractionation demons trated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectivel y, to enkephalin-containing chromaffin granules (secretory vesicles). Immun ofluorescence cytochemistry of chromaffin cells indicated a discrete, punct ate pattern of ACT immunostaining that resembles that of [Met]enkephalin th at is stored in secretory vesicles. Chromatography of adrenal medullary ext racts through DEAE-Sepharose and chromatofocusing resulted in the separatio n of ACT-like proteins I and II that possess different isoelectric points o f 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by SephacryI S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavali n A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K-i,K-app of 35 nM, but ACT-like p rotein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification o f two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granu les indicates that they could interact in vivo. Results from this study sug gest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processi ng in adrenal medulla.