TY - JOUR
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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U2 - 10.1046/j.1432-1327.1998.2540050.x
DO - 10.1046/j.1432-1327.1998.2540050.x
M3 - Article
C2 - 9652393
AN - SCOPUS:0002728435
SN - 1742-464X
VL - 254
SP - 50
EP - 57
JO - FEBS Journal
JF - FEBS Journal
IS - 1
ER -