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-nitrophenyl-p'-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, PPAF-I 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.
authors.affiliation: College of Pharmacy, Pusan National University, Kumjeong Ku, Korea.
authors.forename: S Y
authors.initials: SY
authors.lastname: Lee
authors.forename: T H
authors.initials: TH
authors.lastname: Kwon
authors.forename: J H
authors.initials: JH
authors.lastname: Hyun
authors.forename: J S
authors.initials: JS
authors.lastname: Choi
authors.forename: S I
authors.initials: SI
authors.lastname: Kawabata
authors.forename: S
authors.initials: S
authors.lastname: Iwanaga
authors.forename: B L
authors.initials: BL
authors.lastname: Lee
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D001517:Coleoptera / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D019476:Insect Proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D012697:Serine Endopeptidases; D000581:Amidohydrolases