Isolation and characterization of a novel serine proteinase complexed with alpha 2-macroglobulin from porcine gastric mucosa.

Porcine stomach mucosa was found to contain a 740-kDa protein having endopeptidase activity toward peptide 4-methylcoumaryl-7-amide substrates and low molecular mass peptides. This protein was purified to an apparent homogeneity by a series of chromatographic steps on DEAE-cellulose, Sepharose CL-4B, hydroxylapatite, and fast protein liquid chromatography Mono Q columns. The protein was shown to be a complex of the plasma proteinase inhibitor alpha 2-macroglobulin and a 25-kDa endopeptidase. The enzyme activity was completely inhibited by diisopropyl fluorophosphate, p-amidinophenylmethanesulfonyl fluoride, leupeptin, antipain, bovine pancreatic trypsin inhibitor, soybean trypsin inhibitor, and ovomucoid, indicating that the entrapped enzyme is a serine proteinase. The proteinase could be released from alpha 2-macroglobulin by mild acid treatment and the released enzyme showed activity toward protein substrates. Substrate specificity studies using synthetic and peptide substrates indicated that the enzyme preferentially hydrolyzes Arg-X bonds and, to a much lesser extent, Lys-X bonds, and is apparently distinct from thrombin, kallikrein, plasmin, and other trypsin-like proteinases so far reported including tryptase. Thus, the present enzyme is thought to be a novel type of serine proteinase. The proteinase associated with alpha 2-macroglobulin was also found in porcine intestinal mucosa, but not in plasma.