In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol- oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae

So Young Lee, Tae Hyuk Kwon, Ji Hoon Hyun, 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

104 引用 (Scopus)

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Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro- PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl- phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p- nitrophenylp'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2- terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAFI converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF- II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.

元の言語英語
ページ(範囲)50-57
ページ数8
ジャーナルEuropean Journal of Biochemistry
254
発行部数1
DOI
出版物ステータス出版済み - 5 15 1998

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Monophenol Monooxygenase
Hemolymph
Larva
Chemical activation
amidase
Serine Proteases
Molecular mass
Fluorescent Dyes
Polyacrylamide Gel Electrophoresis
In Vitro Techniques
Proteins
Antibody Affinity
Substrates
Immunoblotting
Gel Chromatography
Insects
Gels

All Science Journal Classification (ASJC) codes

  • Biochemistry

これを引用

In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol- oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. / Lee, So Young; Kwon, Tae Hyuk; Hyun, Ji Hoon; Choi, Jae Sue; Kawabata, Shun-Ichiro; Iwanaga, Sadaaki; Lee, Bok Luel.

:: European Journal of Biochemistry, 巻 254, 番号 1, 15.05.1998, p. 50-57.

研究成果: ジャーナルへの寄稿記事

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title = "In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol- oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae",
abstract = "Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro- PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl- phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p- nitrophenylp'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2- terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAFI converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF- II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.",
author = "Lee, {So Young} and Kwon, {Tae Hyuk} and Hyun, {Ji Hoon} and Choi, {Jae Sue} and Shun-Ichiro Kawabata and Sadaaki Iwanaga and Lee, {Bok Luel}",
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T1 - In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol- oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae

AU - Lee, So Young

AU - Kwon, Tae Hyuk

AU - Hyun, Ji Hoon

AU - Choi, Jae Sue

AU - Kawabata, Shun-Ichiro

AU - Iwanaga, Sadaaki

AU - Lee, Bok Luel

PY - 1998/5/15

Y1 - 1998/5/15

N2 - Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro- PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl- phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p- nitrophenylp'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2- terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAFI converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF- II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.

AB - Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro- PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl- phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p- nitrophenylp'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2- terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAFI converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF- II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.

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