Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae

Tae Hyuk Kwon, So Young Lee, Ji Hye Lee, Jae Sue Choi, Shun Ichiro Kawabata, Sadaaki Iwanaga, Bok Luel Lee

研究成果: ジャーナルへの寄稿記事

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Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.

元の言語英語
ページ(範囲)90-97
ページ数8
ジャーナルMolecules and cells
7
発行部数1
出版物ステータス出版済み - 2 28 1997

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Monophenol Monooxygenase
Hemolymph
Larva
Insects
Dextran Sulfate
Chromatography
Polyacrylamide Gel Electrophoresis
pro-phenoloxidase
Enzyme Precursors
Phenol
Proteolysis
Gel Chromatography
Proteins
Molecular Weight
High Pressure Liquid Chromatography
Amino Acids
Enzymes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

これを引用

Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae. / Kwon, Tae Hyuk; Lee, So Young; Lee, Ji Hye; Choi, Jae Sue; Kawabata, Shun Ichiro; Iwanaga, Sadaaki; Lee, Bok Luel.

:: Molecules and cells, 巻 7, 番号 1, 28.02.1997, p. 90-97.

研究成果: ジャーナルへの寄稿記事

Kwon, Tae Hyuk ; Lee, So Young ; Lee, Ji Hye ; Choi, Jae Sue ; Kawabata, Shun Ichiro ; Iwanaga, Sadaaki ; Lee, Bok Luel. / Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae. :: Molecules and cells. 1997 ; 巻 7, 番号 1. pp. 90-97.
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title = "Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae",
abstract = "Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.",
author = "Kwon, {Tae Hyuk} and Lee, {So Young} and Lee, {Ji Hye} and Choi, {Jae Sue} and Kawabata, {Shun Ichiro} and Sadaaki Iwanaga and Lee, {Bok Luel}",
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T1 - Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae

AU - Kwon, Tae Hyuk

AU - Lee, So Young

AU - Lee, Ji Hye

AU - Choi, Jae Sue

AU - Kawabata, Shun Ichiro

AU - Iwanaga, Sadaaki

AU - Lee, Bok Luel

PY - 1997/2/28

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N2 - Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.

AB - Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.

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