The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells.

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.

Molecular cloning demonstrates structural features of homologous bovine prohormone convertases 1 and 2.

PC1 and PC2 (prohormone convertase) represent neuroendocrine members of the mammalian subtilisin-like family of proprotein convertases. The goal of this study was to compare the primary sequence motifs of bovine PC1 and PC2 with those of homologs from other mammalian species to establish the structural basis for PC1 and PC2 activities in bovine that resemble other mammalian homologs. Molecular cloning from bovine adrenal medulla resulted in the isolation of cDNAs for bovine PC1 and PC2 with highly conserved primary sequences with respect to signal sequence, prosegment, catalytic domain, and P domain.

Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for dithiothreitol-sensitive inhibition of elastase.

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, suggesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory activity. To test these predicted properties, mutagenesis and chemical modification of recombinant endopin 2 were performed to examine the influence of dithiothreitol (DTT), a reducing agent, on endopin 2 activity.

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1.

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules.

Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

An alpha1-antichymotrypsin-like serpin has been implicated in Alzheimer's disease (AD) based on immunochemical detection of alpha1-antichymotrypsin (ACT) in amyloid plaques from the hippocampus of AD brains. The presence of neuroendocrine isoforms of ACTs and reported variations in human liver ACT cDNA sequences raise the question of the molecular identity of ACT in brain. In this study, direct reverse transcription-polymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases.