Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology(2008)
摘要
Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA). Both Mg2+ and Cu2+ stimulated PO activity when compared with controls. The β-sheet content of PPO treated with Mg2+ and Cu2+ increased significantly (P<0.05). The purified PPO has magnesium level of 5.674±2.294 µg/mg and copper level of 1.257±0.921 µg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.
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关键词
Kinetics,Metal ion,Ostrinia furnacalis,Prophenoloxidase,Secondary structure
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