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.
Uchino
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Sakurai
Y
Y
Nishigai
M
M
Takahashi
T
T
Arakawa
H
H
Ikai
A
A
Takahashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013917:Thrombin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000302:isolation & purification* / Q000378:metabolism*
D006882:Hydroxyapatites; D046911:Macromolecular Substances; D009842:Oligopeptides; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D017886:Durapatite; D007610:Kallikreins; D012697:Serine Endopeptidases; D013917:Thrombin; D005341:Fibrinolysin