P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function

AbstractHelicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30–40°C, respectively. Kinetic studies indicated that this enzyme had a Km value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest

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Not AvailablePhenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hübner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDS–PAGE, MALDI–TOF MS and LC–ESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit‐1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L‐Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.Not Availabl