title: Purification and characterization of a digestive alkaline protease from the larvae of Spilosoma obliqua.
abstract: A digestive protease from Spilosoma obliqua (Lepidoptera: Arctiidae) fifth instar larval guts was purified and characterized. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and hemoglobin-sepharose affinity chromatography. The purification procedure resulted in a 37-fold increase in the specific activity of the protease. Protease thus obtained was found to be electrophoretically pure under native and denaturing conditions. The purified protease had a molecular mass of 90 kDa as determined by gel filtration, and a pH optimum of 11.0. The purified protease optimally hydrolyzed casein at 50 degrees C. A Km of 2 x10(-6) M was obtained using BApNA as a substrate for the purified alkaline protease. The ability of S. obliqua protease and bovine trypsin to hydrolyze various synthetic substrates (BApNA, BAEE, and BAME), and the inhibition patterns of S. obliqua and bovine trypsin with "classical" trypsin inhibitors are also reported.
authors.affiliation: Department of Pharmaceutical Sciences, Program in Molecular Toxicology, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. adil.anwar@uchsc.edu
authors.forename: Adil
authors.initials: A
authors.lastname: Anwar
authors.forename: M
authors.initials: M
authors.lastname: Saleemuddin
mesh_terms: D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D011480:Protease Inhibitors; D001586:Benzoylarginine Nitroanilide; D010450:Endopeptidases