Purification and Characterization of a Vasoactive Intestinal Polypeptide-degrading Endoprotease from Porcine Antral Mucosal Membranes

Abstract

A neutral endoprotease was isolated from porcine antral mucosa and purified to homogeneity as examined by SDS-polyacrylamide gel electrophoresis (PAGE). Throughout the purification, t-butyloxycarbonyl-Arg-Val-Arg-Arg-4- methylcoumaryl-7-amide (MCA) was used as a substrate, which was found to be hydrolyzed specifically by the enzyme at the Arg-Arg bond. Unexpectedly, however, the enzyme was also found to hydrolyze vasoactive intestinal polypeptide (VIP) fairly specifically and more efficiently when various neuropeptides and related peptides were examined as substrates. It could degrade VIP by cleaving three peptide bonds not containing an arginine residue(s) with Km = 7.7 x 10(-6) M and kcat/Km = 7.4 x 10(6) M-1 s-1 (at pH 7.6 in the presence of 0.1% Lubrol PX), whereas only secretin, substance P, and a few others were hydrolyzed at much slower rates among the various peptides examined. Both activities toward the MCA substrate and VIP behaved in parallel throughout the purification procedures and showed essentially the same pH optimum and susceptibility toward various inhibitors and detergents. Therefore, both activities are thought to be due to the same enzyme. This endoprotease required 0.001% or a higher concentration of a detergent such as Lubrol PX or Triton X-100 for its maximal activity. Its optimum pH was about 7.5 and the molecular weight was estimated to be approximately 37,000 by SDS-PAGE. This enzyme was strongly inhibited by serine protease inhibitors such as diisopropyl-fluorophosphate and phenylmethanesulfonyl fluoride. It was also inhibited by p-chloromercuribenzoic acid, but not by some other cysteine protease inhibitors. Therefore, the enzyme appears to be most likely a kind of serine protease although its possibility as a cysteine protease cannot be completely excluded. Analysis of its cleavage specificity toward various oligopeptides indicated the possibility that the protease might recognize a specific amino acid sequence(s) and/or conformation in the vicinity of the cleavage site of the target peptide. Various characteristics of the endoprotease suggest that it is a novel membrane-bound neuropeptide-degrading endoprotease fairly specific for VIP.

Purification and characterization of a detergent‐requiring membrane‐bound metalloendopeptidase from porcine brain

“…It seems difficult to explain the cleavage spepcificity only based on the amino acid residues at the cleavage sites. The enzyme might recognize a conformation and/or a certain subsite structure in the vicinity of the cleavage sites of the target peptides, as previously suggested for a vasoactive intestinal polypeptide‐degrading endopeptidase from porcine antral mucosal membranes [17,18].…”

Enzymic properties of recombinant BACE2

“…It was previously shown that the inhibition of proteolytic activity by metal ions could be nonspecific. For example, E. coli leader peptidase is inhibited nonspecifically by Hg 2+ and Cu 2+ ions (60% inhibition [31]); an endoprotease from porcine antral mucosal membranes is inhibited by Fe 2+ , Cu 2+ , Zn 2+ , and Hg 2+ ions (100% inhibition [32]), among others [33,34]. Therefore, it is speculated that the inhibition of BACE2 by the metal ions is also nonspecific.…”