Bozić Natasa, Ivanović Jelisaveta, Nenadović Vera, Bergström Jörgen, Larsson Thomas, Vujcić Zoran
Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy, Studentski trg 12-16, 11000 Belgrade, Serbia.
Comp Biochem Physiol B Biochem Mol Biol. 2008 Mar;149(3):454-62. doi: 10.1016/j.cbpb.2007.11.006. Epub 2007 Nov 24.
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.
对暗黑鳃金龟幼虫中肠的主要亮氨酰氨肽酶（LAP）进行了纯化和特性鉴定。通过对中肠粗提物的纯化，LAP的比活性提高了292倍。纯化后的酶最适pH为7.5（最适pH范围7.0 - 8.5），优先水解活性位点含有疏水氨基酸的对硝基苯胺，对亮氨酸对硝基苯胺（LpNA）的V(max)/K(M)比值最高。在测试的多种抑制剂中，最有效的是1,10 - 菲咯啉（K(i)值为0.12 mM）和半胱氨酸（K(i)值为0.31 mM），而乙二醇双四乙酸（EGTA）刺激LAP活性。锌离子（Zn(2+)）、镁离子（Mg(2+)）和锰离子（Mn(2+)）对LAP活性均表现出双峰效应（低浓度时激活，高浓度时抑制）。纯化后的LAP（经Superose 6柱凝胶过滤后）分子量为400 kDa，等电点为6.2。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）显示一条67 kDa的条带，表明该酶是六聚体。使用电喷雾串联质谱（ESI/MS - MS）分析从蛋白条带中获得了六个肽序列。将获得的肽序列与EMBL - EBI序列分析工具箱和BLASTP数据库进行比较，结果表明与其他昆虫氨肽酶具有高度同源性。
