Purification and characterization of a novel membrane-bound arginine-specific serine proteinase from porcine intestinal mucosa.
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 non-reducing 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.
Tsuchiya
Y
Y
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Takahashi
T
T
Sakurai
Y
Y
Iwamatsu
A
A
Takahashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D008565:Membrane Proteins; D001120:Arginine; D012697:Serine Endopeptidases