Purification and characterization of alpha 1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing.

Evidence is presented showing that alpha 1-antichymotrypsin (ACT) inhibits a novel prohormone thiol protease (PTP) involved in processing the enkephalin precursor. Colocalization of ACT immunoreactivity with PTP within isolated secretory vesicles of bovine adrenal medulla and pituitary indicated that endogenous ACT could regulate PTP in vivo. The endogenous 60 kDa bovine ACT (bACT)-like protein was purified from pituitary by chromatography on DEAE-Sepharose, chromatofocusing, butyl-Sepharose, and Sephacryl S-200. Characterization showed that the bACT-like protein was a potent inhibitor of PTP (Ki,app value of 2.2 nM) as well as an effective inhibitor of chymotrypsin (Ki,app value of 2.3 nM). Furthermore, the bACT-like protein formed sodium dodecyl sulfate-stable complexes with chymotrypsin, which is typical of serpin protease inhibitors. Importantly, PTP formed sodium dodecyl sulfate-stable complexes with human ACT, suggesting that PTP's cleavage specificity may resemble the reactive center of ACT. PTP cleavage of enkephalin-containing peptides at the NH2-terminal side of paired basic residues (Lys-Arg, Arg-Arg, Lys-Lys), flanking the COOH terminus of (Met)enkephalin (Tyr-Gly-GLy-Phe-Met), indicates methionine at the P1 position. PTP cleavage of peptide-methylcoumarin amide and peptide-p-nitroanilide substrates demonstrated specificity for paired basic and monobasic residues, as well as a role for methionine in PTP's cleavage site. These results showing PTP's ability for processing at a methionine residue which resembles the P1 specificity of ACT are compatible with inhibition of PTP by ACT. These findings are the first demonstration of the involvement of a protease inhibitor in neuropeptide precursor processing. The known developmental regulation of ACT in brain and significant amounts of ACT in amyloid plaques of Alzheimer's disease suggest a possible role for PTP in the maturation of peptidergic neurons.