Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis

dc.creatorOliveira, M.G.A.
dc.creatorSimone, S.G. De
dc.creatorXavier, L.P.
dc.creatorGuedes, R.N.C.
dc.date2018-04-27T14:42:40Z
dc.date2018-04-27T14:42:40Z
dc.date2004-10-31
dc.date.accessioned2023-09-27T20:55:09Z
dc.date.available2023-09-27T20:55:09Z
dc.identifier1096-4959
dc.identifierhttps://doi.org/10.1016/j.cbpc.2004.10.018
dc.identifierhttp://www.locus.ufv.br/handle/123456789/19221
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8950835
dc.descriptionTrypsin-like proteases from the midgut of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris–HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates l-BApNA and N-α-p-tosyl-l-Arg methyl ester (l-TAME). Higher activity was observed at pH 8.5 and 35 °C when using l-BApNA as substrate and at pH 8.0 and 30 °C when using l-TAME. Maximum enzyme activity against l-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl-l-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for l-BApNA and 52.5 μM for l-TAME.
dc.formatpdf
dc.formatapplication/pdf
dc.languageeng
dc.publisherComparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
dc.relationVolume 140, Issue 3, Pages 369-380, March 2005
dc.rightsElsevier Inc.
dc.subjectTrypsin
dc.subjectSerine proteases
dc.subjectDigestive proteases
dc.subjectLepidoptera midgut
dc.subjectSubstrate specificity
dc.subjectCleavage site
dc.subjectNoctuidae
dc.subjectProtease inhibition
dc.subjectProtease kinetics
dc.subjectSynthetic peptide hydrolysis
dc.titlePartial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis
dc.typeArtigo


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