Activation of prophenoloxidase A1 by an activating enzyme in Drosophila melanogaster.
An activating enzyme for prophenoloxidase A1 was isolated from pupae of Drosophila melanogaster, and the activation of purified prophenoloxidase A1 with this enzyme was analyzed. The purification included ammonium sulfate fractionation, DEAE-cellulose, Superdex 75, arginine-Sepharose and hydroxyapatite column chromatography. The prophenoloxidase activating enzyme was determined to be a 28.5-kDa protein consisting of a single polypeptide. The kinetics of the activation reactions was unusual in that the final levels of phenoloxidase activity varied depending on the initial concentrations of the activating enzyme, not those of the prophenoloxidase. The activation was effectively suppressed by the inhibitors of trypsin-type serine protease. The protein has amidolytic activity, and Boc-Val-Pro-Arg-MCA was the best substrate among the synthetic substrates examined. The molecular mass of the activated phenoloxidase was smaller than that of the prophenoloxidase, indicating that a 5-kDa peptide was released from the prophenoloxidase by limited proteolysis with the activating enzyme. The cleavage site of prophenoloxidase A1 was shown to be between Arg and Phe at positions 52 and 53.
Chosa
N
N
Biological Laboratory, Faculty of Science, Okayama University of Science, Japan.
Fukumitsu
T
T
Fujimoto
K
K
Ohnishi
E
E
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D012697:Serine Endopeptidases
D004792:Enzyme Precursors; D019476:Insect Proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme; D000581:Amidohydrolases