Abstract
Phenoloxidase (PO) is a key factor in insect immunity. On invasion of microorganisms and pathogens, prophenoloxidase (PPO) changes to its active form, PO. The present study has been conducted to purify and characterize the PO from the haemolymph of desert locust, Schistocerca gregaria (Forskal) following activation of immune system by invasion of bacteria, Bacillus thuringiensis kurstaki (Bt). PO is purified by a combination of ammonium sulfate precipitation, blue sepharose CL-6B and phenyl sepharose CL-4B chromatography yielded a 209.97-fold purity and 54.75% recovery of activity. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) reveals that the molecular weight of the purified PO is 70.154 kDa. The purified PO is characterized in terms of its biochemical and enzymatic properties by using L-DOPA as a specific substrate. Ca2+ and Cu2+ significantly stimulated PO activity when compared with other metals. The PO reaction was strongly inhibited by phenylthiourea and thiourea, moderately inhibited by ethylene diamine tetractic acid (EDTA) and poorly inhibited by ethylene glycol tetraacetic acid (EGTA) and diethyl dithiocarbamate (DTC). Inhibition of PO showed excellent recovery ability by addition of Ca2+ on EGTA-inhibited enzyme. Therefore, PO is most probably a kind of tyrosinase-type Ca2+-containing metalloenzyme. The content of Ca2+ is higher than other trace metal elements. The reactive intermediates yielded by PO with its specific substrate L-DOPA had a broad-spectrum bactericidal activity against Gram +ve bacteria (Bacillus cereus and Staphylococcus aureus) with a greater degree more than Gram-ve bacteria (Escherichia coli and Pseudomonas aeruginosa). From the present study, PO from S. gregaria is most probably a tyrosinase-type calcium-containing mono-phenoloxidase, which functions not only as a catalytic enzyme in melanin production in locusts, but perhaps also as a humoral factor in host defense via melaninization as in other insects. Key words: Schistocerca gregaria, phenoloxidase, purification.
Highlights
The desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) represents a relatively important group of plant-feeding insects
The present study aims to isolate, purify and characterize the components involved in the PO cascade system, and to clarify more information dealing with the physicochemical properties of phenoloxidase of S. gregaria
The total protein content of purified haemolymph after serial purification steps using ammonium sulphate precipitation (NH4)2SO4, 40% saturation followed by affinity chromatography (Blue Sepharose CL-6B chroma-tography and Phenyl Sepharose CL-4B chromatography) were decreased significantly compared with unpurified haemolymph
Summary
The desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) represents a relatively important group of plant-feeding insects Humoral mechanisms that are essentially concerned with the ability of insect to recognize and dispose self from nonself, and involve several physiologically active substances, normally present in the native haemolymph or synthesized after natural infections (Boman and Hultmark, 1987). These substances appear in the haemolymph within a few hours after infection and display a broad spectrum of antimicrobial activity. When insects are infected by microorganisms, PPO activation elicits by microbial cell surface components, such as, lipopolysaccharide (LPS), peptidoglycans, β-1,3glucose (Mo'men et al, 2012), the activities of the haemocytic enzymes, including phenoloxidase are enhanced during the challenge course
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