No abstract

Seventy-four peptide amides of 7-amino-4-methylcoumarine (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBz1)-Pro-Arg-NH-Mec (k,,, = 160 s-', K, = 11 pM, k,,,/K, = 15000000 M-' s-') for human a-thrombin, Z-< Glu-Gly-Arg-NH-Mec (k,,, = 19 s-', K, = 59 pM, k,,,/K, = 320000 M-' s-') for bovine factor Xa, BocGln-Gly-Arg-NH-Mec (k,,, = 5.8 s-', K, = 140 pM, k,,,/K, = 42000) for bovine factor XIIa, Boc-Asp(OBz1)-Ala-Arg-NH-Mec (k,,, = 9.2 s-', K,,, = 120 pM, k,,,/K, = 77000 M-' s -l ) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (k,,, = 29 s-', K, = 230 pM, kcat/Km = 130000 M-' s-') for bovine plasma kallikrein. Moreover, Boc-Glu(OBz1)-Ala-Arg-NH-Mec (k,,, = 46 s-', K, = 370 pM, kcat/Km = 120000 M-' s-'> was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes. The utility of these fluorogenic peptide substrates has been established for the assay of trace amounts of enzymes because of their high sensitivities. Moreover, the specific substrates for a-thrombin, factor Xa, plasma kallikrein and urokinase so far developed by our group [7] are now commercially available.

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, 90Ϫ97]. 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 NH 2 -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 NH 2 -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.

Clones encoding pro-phenol oxidase [pro-

Previously, we identified two pro‐phenol oxidase‐activating factors, named PPAF‐I and PPAF‐II, directly involved in the activation of the purified pro‐phenol oxidase (pro‐PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90−97]. Here, we report molecular cloning of cDNA for PPAF‐I. Based on the sequence of the cloned cDNA, the PPAF‐I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40 193 Da. The 109 amino acid residues preceding the amino‐terminus Ile residue of the mature protein seem to constitute a prepro‐sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28 009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9 % identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro‐segment of PPAF‐I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil‐derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)‐labeled PPAF‐I was specifically produced from the crude preparation of PPAF‐I zymogen by incubation with lipopolysaccharide or 1,3‐β‐glucan, whereas [3H]iPr2P‐labeled PPAF‐I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro‐PO‐activation system in H. diomphalia larvae may proceed with the activation of PPAF‐I zymogen by microbial polysaccharides.