title: Purification and characterization of kallikrein-like serine proteases from rat submandibular glands.
abstract: The purification and characterization of kallikrein-like proteases from rat submandibular glands is described. The proteolytic activity of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME). The purification scheme involved ammonium sulfate precipitation, chromatography on columns of DEAE-Sepharose and Sephadex G-100 and chromatofocusing. Three TAME-hydrolytic activity peaks were eluted from DEAE-Sepharose as unbound fraction (Pool 1), at 125 mM NaCl (Pool 2) and at 250 mM NaCl concentration (Pool 4). Pool 1 further resolved into two protease fractions (1A1 and 1A2), pool 2 into three protease fractions (2A1, 2A2 and 2A3) and pool 4 gave a single major protease peak (4A1) by chromatofocusing on PBE-94. Protease pools 2A2, 2A3, and 4A1 each gave a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 34 kDa, 46 kDa and 46 kDa respectively. Pools 1A1, 1A2, 2A1 and 2a2 gave a single precipitin line with anti-rat glandular kallikrein antibodies. 2A3 and 4A1 did not react with these antibodies. Synthetic substrates DL-val-leu-arg-pNA and Bz-pro-phe-arg-pNA, specific for kallikrein-like proteases, were hydrolyzed preferentially by 2A3 and 4A1 but were poor substrates for 1A1, 1A2, 2A1 and 2A2.
authors.affiliation: Department of Oral Biology, State University of New York, Buffalo 14214.
authors.forename: G S
authors.initials: GS
authors.lastname: Bedi
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000276:immunology; D051381:Rats; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D007610:Kallikreins; D012697:Serine Endopeptidases