الفهرس 
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Abstract
The present investigation has been conducted to describe the purification and characterization of haemolymph phenoloxidase (PO) from desert locust, Schistocerca gregaria, following activation of insect`s immune system by injecting sublethal concentration of the bacterium, Bacillus thuringiensis kurstaki (Bt) into the haemocoel, and to obtain more information dealing with the physicochemical properties of this enzyme and its biological activity. Therefore, the present study covers the following points:
1- Susceptibility of locusts to Bt:
The present study clearly demonstrated that the adults of S. gregaria are resistant to Bt (LC50 = 1.8X 107 cells/ml). This may be due to the nature of diet used in the present study, or it may be due to the powerful defense mechanisms of the target insect. The estimated LC20 was 1.2X106cells/ml, this concentration was found to induce the immune response of locusts and at the same time did not cause high mortality rate. Therefore, this concentration 1.2X106 cells/ml was used as sub-
lethal concentration to investigate the subsequent experiments.
2- Phenoloxidase isolation and purification:
The pre-activation of S. gregaria immune system by Bt-injection, caused a significant increase in PO activity compared with control. The observed formation of PO suggests that; the active enzyme is present at all times but is initially blocked by a dissociable inhibitor that gradually decays, or the enzyme is initially present as an inactive precursor or prophenoloxidase (PPO) that gradually transforms to the active state.
Following bacterial infection and PPO activation, the haemolymph showed a rapid increase in the total haemolymph proteins, such increase may be attributed to the synthesis of new proteins and peptides that enables the insect to oppose a large spectrum of pathogenic microorganisms.
Following hemolymph collection from injected S. gregaria, the PO was purified from the haemolymph of to homogeneity. The finally eluted was 209.97-fold purification with a 54.75% total recovery of activity. The total protein content showed a significant decrease through the serial purification steps, accompanied by a
significant increase in the PO activity compared with those of unpurified sample.
3- Protein electrophoresis:
To reveal the total proteins of the unpurified hemolymph and its subsequent purification steps until reaching the final elution (PO), SDS-PAGE was carried out. Results indicated that the purified PO showed a single polypeptide chain with a molecular weight (MW) of 70.154 kDa from the eluted fractions following purification steps. This indicated that the protocol using the combination of ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography was effective in processing the purification of PO from the haemolymph of S. gregaria.
4- Polyclonal antibody production against purified PO:
Rabbit antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting analysis, by which one band of MW 70.154 kDa corresponding to the purified PO band was detected. These results indicated that S. gregaria PO released in the haemolymph following activation of the immune system of the insect, facilitating the future
determination of the origin of PO in the humoral fluid and the distribution of PO-synthesizing tissues in S. gregaria.
5- Amino acid analysis of the purified PO:
The present investigation indicated 20 types of amino acids using, High-performance liquid chroma-tography (HPLC). In comparison with other insect POs, our results were very similar to that of Locusta migratoria PPO.
6- PO sequencing and searching for sequence homologies in protein databases:
N-terminal amino acid sequence was determined by automatic protein sequencer to further confirm the S. gregaria PO had been purified, 291 amino acid residues have been detected. PO sequence analysis via Basic Local Alignment Search Tool (BLAST) showed that the deduced amino acid sequence has regions of local similarity ranged 82- 50% between S. gregaria PO sequence and other 63 PPO and PO sequences from different insects. There was a high percentage of identity to two members of order Orthoptera (82-80% identity). Identity percentages decreases as comparisons were made to more divergent species in other insect orders.
For further confirmation the Phylogenetic analysis revealed that S. gregaria PO is closely related to order Orthoptera, on the other hand, it was distinct from PPO of other insect orders including (Diptera, Lepidoptera, Hymenoptera, Coleoptera and Hemiptera). The relation-ships between S. gregaria PO and sequence of 63 members in GenBank were obtained from CLASTAL OMEGA, sequence alignments, and recorded that the copper binding sites are conserved in all of the proteins. A putative conserved domain has been observed including Hemocyanin, showed that S. gregaria PO share similarities of 55% with Pediculus humanus corporis hemocyanins.
7- Effect of metal ions on PO activity:
Several metal ions, tested with the purified PO of S. gregaria, showed that PO activity is significantly increased when Ca2+concentration increases, moderately increased in the presence of Cu2+ ions and insignificantly increased with Mg2+, Zn2+ and Mn2+ ions. In the present study, it was found that the content of calcium is higher than other trace metal elements.
8- Inhibition of PO activity:
In order to determine the effect of various inhibitors on the activity of the purified PO from the haemolymph of S. gregaria, different compounds including: thiourea, phenylthiourea, ethylene diamine tetraacetic acid (EDTA), diethyldithiocarbamate (DTC) and ethylene glycol tetraacetic acid (EGTA), were tested. The PO activity was completely inhibited by phenylthiourea and thiourea, moderately inhibited by EDTA and poorly inhibited by and EGTA, DTC. These results suggesting that the binding of some calcium atoms is necessary in the activating the PO of S. gregaria.
The result of recovery test showed that the enzymatic activity of the purified PO greatly inhibits by 20 mM EGTA, restores to its original level by 15mM Ca2+. from these results it may concluded that S. gregaria PO is most probably a kind of calcium-containing metalloenzyme.
9- Enzyme activity and substrate specificity:
In order to investigate the substrate specificity of PO purified from the haemolymph of S. gregaria, different substrates were used; the o-diphenols: 4
methylcatechol, catechol, gallic acid, the L-dihydroxy phenylalanine (L-DOPA), and monophenol tyrosin. The PO from S. gregaria is capable of oxidizing L-DOPA effectively, but fails to oxidize tyrosine. These results implies that this enzyme is most probably a kind of monophenol, tyrosinase-type o-oxidoreductase, not a laccase-type or catechol oxidase-type enzyme.
10- Effect of PO-substrate-derived compounds on bacteria (antibacterial activity):
Preliminary experiments showed that the bacterial growth is not affected by treatment with the purified S. gregaria PO alone. In order to test the effect of PO-substrate-derived compounds on the growth and survival of bacteria, dependent on their growth inhibition rates, Gram +ve bacteria (Bacillus cereus and Staphylococcus aureus) and Gram -ve bacteria (Escherichia coli and Pseudomonas aeruginosa) were incubated with PO with its specific substrate L-DOPA. Results indicated that the reactive intermediates yielded by PO had a broad-spectrum bactericidal activity. Gram –ve bacteria were most resistant than Gram +ve bacteria. These results established that PPO activation system is an integral component of the insect defense system involving a
multitude of enzymes which immobilizes and kills invading bacteria. These results were confirmed by the findings of inhibition zone assays, and may attribute such reduction in the bacterial growth to the synthesis of new proteins associated with immune activation.