title: Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae.
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. Zn(2+), Mg(2+) and Mn(2+) 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.
authors.affiliation: Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy, Studentski trg 12-16, 11000 Belgrade, Serbia. nbozic@chem.bg.ac.yu
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
authors.forename: Jörgen
authors.initials: J
authors.lastname: Bergström
authors.forename: Thomas
authors.initials: T
authors.lastname: Larsson
authors.forename: Zoran
authors.initials: Z
authors.lastname: Vujcić
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002413:Cations, Divalent / Q000494:pharmacology; D001517:Coleoptera / Q000187:drug effects / Q000201:enzymology*; D004064:Digestive System / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007525:Isoelectric Focusing; D007700:Kinetics; D007814:Larva / Q000187:drug effects / Q000201:enzymology; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D007931:Leucyl Aminopeptidase