Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae
Authorized Users Only
2008
Authors
Božić, NatašaIvanovic, Jefisaveta
Nenadovic, Vera
Bergstroem, Joergen
Larsson, Thomas
Vujčić, Zoran
Article (Published version)
Metadata
Show full item recordAbstract
The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V-max /K-M ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1, 10-phenanthroline having a K-i value of 0.12 mM and cysteine with K-i value of 0.31 mM, while EGTA stimulated LAP activity. Zn2+, Mg2+ and Mn2+ all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggestin...g that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases. (C) 2007 Elsevier Inc. All rights reserved.
Keywords:
leucyl aminopeptidase / midgut / isoform / cerambycid beetle / Morimus funereus / xylophagous larvaeSource:
Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology, 2008, 149, 3, 454-462Publisher:
- Elsevier Science Inc, New York
Funding / projects:
- Interakcije prirodnih proizvoda i njihovih analoga sa proteinima i nukleinskim kiselinama (RS-MESTD-MPN2006-2010-142026)
DOI: 10.1016/j.cbpb.2007.11.006
ISSN: 1096-4959
PubMed: 18155948
WoS: 000253576300007
Scopus: 2-s2.0-39049120322
Collections
Institution/Community
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Božić, Nataša AU - Ivanovic, Jefisaveta AU - Nenadovic, Vera AU - Bergstroem, Joergen AU - Larsson, Thomas AU - Vujčić, Zoran PY - 2008 UR - https://cherry.chem.bg.ac.rs/handle/123456789/919 AB - The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V-max /K-M ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1, 10-phenanthroline having a K-i value of 0.12 mM and cysteine with K-i value of 0.31 mM, while EGTA stimulated LAP activity. Zn2+, Mg2+ and Mn2+ all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases. (C) 2007 Elsevier Inc. All rights reserved. PB - Elsevier Science Inc, New York T2 - Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology T1 - Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae VL - 149 IS - 3 SP - 454 EP - 462 DO - 10.1016/j.cbpb.2007.11.006 ER -
@article{ author = "Božić, Nataša and Ivanovic, Jefisaveta and Nenadovic, Vera and Bergstroem, Joergen and Larsson, Thomas and Vujčić, Zoran", year = "2008", abstract = "The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V-max /K-M ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1, 10-phenanthroline having a K-i value of 0.12 mM and cysteine with K-i value of 0.31 mM, while EGTA stimulated LAP activity. Zn2+, Mg2+ and Mn2+ all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases. (C) 2007 Elsevier Inc. All rights reserved.", publisher = "Elsevier Science Inc, New York", journal = "Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology", title = "Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae", volume = "149", number = "3", pages = "454-462", doi = "10.1016/j.cbpb.2007.11.006" }
Božić, N., Ivanovic, J., Nenadovic, V., Bergstroem, J., Larsson, T.,& Vujčić, Z.. (2008). Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae. in Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology Elsevier Science Inc, New York., 149(3), 454-462. https://doi.org/10.1016/j.cbpb.2007.11.006
Božić N, Ivanovic J, Nenadovic V, Bergstroem J, Larsson T, Vujčić Z. Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae. in Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology. 2008;149(3):454-462. doi:10.1016/j.cbpb.2007.11.006 .
Božić, Nataša, Ivanovic, Jefisaveta, Nenadovic, Vera, Bergstroem, Joergen, Larsson, Thomas, Vujčić, Zoran, "Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae" in Comparative Biochemistry and Physiology. B: Biochemistry and Molecular Biology, 149, no. 3 (2008):454-462, https://doi.org/10.1016/j.cbpb.2007.11.006 . .