Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut.
We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of approximately 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50-60 degrees C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca(+2), Mn(+2) and Mg(+2) somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.
Lomate
Purushottam R
PR
Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University Aurangabad, Maharashtra State, India.
Hivrale
Vandana K
VK
D000818:Animals; D001413:Bacillus thuringiensis; D001427:Bacterial Toxins / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology* / Q000378:metabolism*; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043384:Glutamyl Aminopeptidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000033:anatomy & histology / Q000201:enzymology; D007915:Lepidoptera / Q000033:anatomy & histology / Q000201:enzymology* / Q000254:growth & development; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D008670:Metals; D043384:Glutamyl Aminopeptidase