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学術誌査読

123 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)50-57
ページ数8
ジャーナルEuropean Journal of Biochemistry
254
1
DOI
出版ステータス出版済み - 5月 15 1998

!!!All Science Journal Classification (ASJC) codes

  • 生化学

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