A link between blood coagulation and prophenol oxidase activation in arthropod host defense.
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Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade. (+info)
An improved method of light-induced pigmentation.
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An improved procedure was developed whereby a primary light signal can be intensified and made visible by activation of a pre-tyrosinase (pre-phenoloxidase) enzyme [isolated from silkworm (Bombyx mori)] by alpha-chymotrypsin; this activation results from the light-activated conversion of the inactive cis-cinnamoyl-alpha-chymotrypsin. (+info)
Expression of prophenoloxidase mRNA during silkworm hemocyte development.
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Two clones encoding different prophenoloxidase isoforms were amplified by polymerase chain reaction of RNA from the hemocytes of an experimental strain of Bombyx mori. The nucleotide sequences of the clones and the deduced amino acid sequences were confirmed to be nearly identical to those of the orthologous clones previously obtained from a commercial race of B. mori. Northern blot hybridization using these clones as probes demonstrated that the prophenoloxidase mRNA in the hemocytes is expressed in a stage-specific manner during the final larval instar and pupal stage, showing a peak one day before pupation in males and on the day of pupation in females. A sexual difference was also observed when the content of prophenoloxidase protein in the hemolymph (including hemocytes) was measured by an enzyme-linked immunosorbent assay. (+info)
Inhibitory effects of plant polyphenoloxidase on colonization factors of Streptococcus sobrinus 6715.
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Exogenously added polyphenoloxidase (EC 1.14.18.1), an enzyme which oxidizes tyrosine residues and is commonly found in many dietary components, abolished the aggregation of Streptococcus sobrinus 6715 by high-molecular-weight dextran. The enzyme decreased glucan-binding lectin and/or glucosyltransferase I activities. (+info)
Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory.
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The inducible expression of polyphenol oxidase (PPO), a presumed antiherbivore enzyme, was examined in hybrid poplar (Populus trichocarpa x Populus deltoides). Following mechanical wounding simulating insect damage, PPO activity increased dramatically in wounded and unwounded leaves on wounded plants beginning at 24 and 48 h, respectively. A hybrid poplar PPO cDNA was isolated and its nucleotide sequence determined. On northern blots, PPO transcripts were detected within 8 h of wounding, and reached peak levels at 16 and 24 h in wounded and unwounded leaves, respectively. Methyl jasmonate spray and feeding by forest tent caterpillar also induced PPO expression. The induction of PPO was strongest in the youngest four leaves, which were generally avoided by caterpillars in free feeding experiments. This wound- and herbivore-induced expression of PPO in hybrid poplar supports the defensive role of this protein against insect pests. (+info)
A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.
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Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-kDa protein was cleaved to a 35-kDa protein. RNA blot hybridization revealed that expression of the 45-kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge. (+info)
Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration.
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Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration. (+info)
A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.
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Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus. (+info)