Abstract
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.
Highlights
KDa proteinhavingendopeptidase activity toward the proteolytic processing of such peptides have yet been peptide 4-methylcoumaryl-7-amidesubstrates and low identified
Not thesame a2-macroglobulin-proteinasceomplex is present in the mucosa of the porcine intestine, a partial purification experiment was conducted as described inthe Miniprint section
In the present study we have isolated from porcine stomach mucosa a high molecular weight protein
Summary
Enzyme activities were determinedat pH 9.0 with 50 pM substrates as described under “ExperimentalProcedures” and are expressed as Association of a Serine Proteinase with a2-Macroglobulin- percent of activity toward Boc-Gln-Arg-Arg-MCA. The purified protein Boc-Gln-Gly-Arg-MCA cross-reacted with goat anti-human a2-macroglobulin anti- Boc-Gln-Ala-Arg-MCA. Proteinase Complex-The results shown in Fig. 7 suggest that Suc-Ala-Ala-Pro-Phe-MCA 0 the proteinase is dissociable from the a2-macroglobulin-pro- Suc-Leu-Leu-Val-Tyr-MCA 0. - 0 the activity toward Boc-Gln-Arg-Arg-MCA substrate, but re- ’Not tested. Strong inhibition by soybean trypsin inhibitorand ovomucoidwas observed only afterthe purified protein was treated at pH 3, indicating again the release of the proteinase from az-macroglobulin by thistreatment. These results clearly indicate that the enzyme is a trypsinlike serine proteinase
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