Purification and characterization of a novel membrane-bound arginine-specific serine proteinase from porcine intestinal mucosa.
1994
Tsuchiya Y. | Takahashi T. | Sakurai Y. | Iwamatsu A. | Takahashi K.
A novel membrane-bound serine proteinase has been purified from the microsomal membranes of porcine intestinal mucosa. It was solubilized from the microsomal membrane fraction with 1% sodium deoxycholate, then purified by a series of column chromatographic steps on DE52, butyl-Toyopearl Bio-Gel P-150, Mono Q, and benzamidine-Sepharose in the presence of 0.02% Lubrol PX. Its molecular mass was estimated to be 50 kDa both by SDS-polyacrylamide gel electrophoresis under nonreducing conditions and by gel filtration, and to be 32 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions, suggesting that the enzyme may exist as a homodimer in which two subunits are linked by disulfide bond(s). It had a pH optimum at around 9 and did not require Ca2+ for activity. It cleaved several peptide 4-methylcoumaryl-7-amide substrates almost exclusively after arginine residues, the best substrate among those tested being t-butyloxycarbonyl-Gln-Ala-Arg-4-methylcoumaryl-7-amide. Various neuropeptides were also cleaved by this enzyme after arginine, mainly between paired basic amino acid residues, Arg-Arg or Arg-Lys. Activity toward protein substrates was scarcely detected. Further, its partial amino acid sequences were highly homologous, but not identical, with those of trypsin-type serine proteinases. These results indicate that the present enzyme is a novel arginine-specific trypsin-like endopeptidase, possibly involved as a processing proteinase in the production of certain gastrointestinal neuropeptides or peptide hormones from their precursors, or their specific degradation.
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