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Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis.
Comp Biochem Physiol B Biochem Mol Biol. 2005 Mar; 140(3):369-80.CB

Abstract

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (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-alpha-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-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees 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-alpha-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 microM for L-TAME.

Authors+Show Affiliations

Departamento de Bioquímica e Biologia Molecular, Instituto de Biotecnologia Aplicada a Agropecuária (BIOAGRO), Universidade Federal de Viçosa, Viçosa, MG 36571-000, Brazil. malmeida@ufv.brNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15694584

Citation

Oliveira, M G A., et al. "Partial Purification and Characterization of Digestive Trypsin-like Proteases From the Velvet Bean Caterpillar, Anticarsia Gemmatalis." Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, vol. 140, no. 3, 2005, pp. 369-80.
Oliveira MG, De Simone SG, Xavier LP, et al. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol B Biochem Mol Biol. 2005;140(3):369-80.
Oliveira, M. G., De Simone, S. G., Xavier, L. P., & Guedes, R. N. (2005). Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 140(3), 369-80.
Oliveira MG, et al. Partial Purification and Characterization of Digestive Trypsin-like Proteases From the Velvet Bean Caterpillar, Anticarsia Gemmatalis. Comp Biochem Physiol B Biochem Mol Biol. 2005;140(3):369-80. PubMed PMID: 15694584.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. AU - Oliveira,M G A, AU - De Simone,S G, AU - Xavier,L P, AU - Guedes,R N C, PY - 2004/06/28/received PY - 2004/10/29/revised PY - 2004/10/31/accepted PY - 2005/2/8/pubmed PY - 2005/7/9/medline PY - 2005/2/8/entrez SP - 369 EP - 80 JF - Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology JO - Comp Biochem Physiol B Biochem Mol Biol VL - 140 IS - 3 N2 - Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (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-alpha-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-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees 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-alpha-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 microM for L-TAME. SN - 1096-4959 UR - https://www.unboundmedicine.com/medline/citation/15694584/Partial_purification_and_characterization_of_digestive_trypsin_like_proteases_from_the_velvet_bean_caterpillar_Anticarsia_gemmatalis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-4959(04)00359-8 DB - PRIME DP - Unbound Medicine ER -