title: Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae.
abstract: Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia.
authors.forename: Nikola
authors.initials: N
authors.lastname: Loncar
authors.forename: Zoran
authors.initials: Z
authors.lastname: Vujcić
authors.forename: Natasa
authors.initials: N
authors.lastname: Bozić
authors.forename: Jelisaveta
authors.initials: J
authors.lastname: Ivanović
authors.forename: Vera
authors.initials: V
authors.lastname: Nenadović
mesh_terms: D000818:Animals; D001550:Benzamidines / Q000737:chemistry; D001517:Coleoptera / Q000201:enzymology* / Q000254:growth & development; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D007814:Larva / Q000201:enzymology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D013696:Temperature
chemical_list: D001550:Benzamidines; D019476:Insect Proteins; D015842:Serine Proteinase Inhibitors; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases; C032157:benzamidine