Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

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• 作者：Nataš ; a Bož ; ić ; Jelisaveta Ivanović ; Vera Nenadović ; Jö ; rgen Bergströ ; m ; Thomas Larsson ; Zoran Vuj&#x10d ; ić
• 关键词：Leucyl aminopeptidase ; Midgut ; Isoform ; Cerambycid beetle ; Morimus funereus ; Xylophagous larvae
• 刊名：Comparative Biochemistry and Physiology, Part B
• 出版年：2008
• 出版时间：March 2008
• 年：2008
• 卷：149
• 期：3
• 页码：454-462
• 全文大小：488 K

文摘

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.