Abstract
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.
Highlights
From the $Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tbk.yo 113, Japan and llCentral Laboratories for Key ?kchnology, Kirin Brewery Co
Intestine contains various kindosf proteinases, and some of such enzymes,with special attention tomembrane-bound prothem are known to be involved in digestion of food proteins. teinases, and here we report the complete purification and Ontheotherhand,protein digestion intheintestineis characterization of a novel arginine-specific trypsin-like enthought to be regulated by various kinds of gastrointestinal dopeptidase obtainedfrom the microsomal membrane fraction peptides
As described in the preceding section, over 130,000-foldpurification was needed to obtain the pure enzyme from the microsomal membranes solubilized by sodium deoxycholate
Summary
'ND, not determined, because of the high concentration of ammonium sulfate. 100 63 25 32 21 13 phate (DFP),trypsininhibitors from soybean and bovine pancreas, tion wavelength at 370 nm and an emission wavelength a t 460 nm. Fractions from four runs were pooled and combined, and to this fraction The enzyme on PVDF membranes was reduced and S-carboxymethylwas added ammonium sulfate to 20% saturation. This sample was ated, incubated with Acromobacter protease I (lysylendopeptidase) directly applied to a butyl-Toyopearl column (2.6 x 28 cm) equilibrated and endopeptidase AspN, respectively. Elution was performed with 0.1 M borate-Na,CO, buffer, pH 8.0, et al [28] using the BCA reagent with bovine serumalbumin as a containing 0.2 M NaCl a t a flow rate of 20 mVh. The pooled fraction was standard. Dialyzed against 20 mM Tris-HC1 buffer,pH 8.0, and applied to a FPLC Mono Q HR5/5column (Pharmacia Biotech Inc.) followedby linear
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