Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae.
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 (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA). Both Mg(2+) and Cu(2+) stimulated PO activity when compared with controls. The beta-sheet content of PPO treated with Mg(2+) and Cu(2+) increased significantly (P<0.05). The purified PPO has magnesium level of 5.674+/-2.294 microg/mg and copper level of 1.257+/-0.921 microg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.
Feng
Congjing
C
Department of Plant Protection, College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, Jiangsu Province, China. fengcj@yzu.edu.cn
Song
Qisheng
Q
Lü
Wenjing
W
Lu
Jianfeng
J
D000595:Amino Acid Sequence; D000818:Animals; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006458:Hemolymph / Q000201:enzymology; D007526:Isoelectric Point; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008670:Metals / Q000032:analysis / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary / Q000187:drug effects; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D008670:Metals; D021122:Protein Subunits; C038451:pro-phenoloxidase; D004156:Catechol Oxidase