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Giant hornet (Vespa mandarinia) venomous phospholipases
Toxicon 38 (2000) 1803±1816 www.elsevier.com/locate/toxicon
Giant hornet (Vespa mandarinia ) venomous phospholipases The puri®cation, characterization and inhibitory properties by biscoclaurine alkaloids Takashi Abe a,*, Masato Sugita a, Tsuyoshi Fujikura a, Jiro Hiyoshi a, Michinori Akasu b a
Hornet Research Group, The Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan b Research laboratory, Kaken Shoyaku Pharmaceutical Co.3-37-10, Shimorenjaku, Mitaka, Tokyo 181-0013, Japan Received 6 July 1999; accepted 8 February 2000
Abstract Two species of giant hornet phospholipase B (PLB), a and b, were puri®ed from the venom of Vespa mandarinia. The puri®cation procedure was simpli®ed by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB a and b were 29.5 and 26.0 kDa, respectively. The isoelectric point of a and b enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40±608C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The Km values of a and b enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic speci®city for L-a-phosphatidylcholine-b-oleoil-g-palmitoil, the Km values of a enzyme for g-palmitoil and b-oleoil residues were 0.528 and 1.392 mM, respectively. While the Km values of b enzyme for g-palmitoil and b-oleoil residues were 7.91 and 2.68 mM, respectively. Both a and b enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of a enzyme was competitively inhibited, but b enzyme was uncompetitive.
* Corresponding author. Tel.: +81-48-467-9527; fax: +81-48-462-1552. E-mail address: [email protected] (T. Abe). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 1 0 9 - 4
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Cepharanthine also inhibited noncompetitively PLA2s of bovine pancreas, bee venom and Naja mossambica mossambica. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phospholipase B; Biscoclaurine alkaloids; Cepharanthine; Hornet; Venom; Vespa mandarinia
1. Introduction Venomous PLS found in poisonous animals, especially PLA2s in snake venoms, are widely studied in molecular biology and pharmacology (Kini, 1997). While, in hymenoptera venoms, wasp and hornet venomous PLs are characterized as PLA1, PLA2 and PLB, despite that of bee, Apis mellifera, venom is PLA2 (Habermann and Neumann, 1957). In hornet species of Vespa xanthoptera (Takasaki and Fukumoto, 1989) and V. basalis (Ho and Ko, 1988), only the PLs were puri®ed and characterized. Therefore, we have not yet reached the knowledge of the common properties of the hornet venomous PLs which also have multiple functions, and is one of the most toxic components in the venoms. On the other hand, we still have serious hornet stung injuries, especially by giant hornets, V. mandarinia, in Japan. Over 40 of those injured have been killed by wasp stings each year. However, there have not been better therapy to cure the sting injuries. Especially for the demand to save lives of bee keepers, lumbermen, farmers and gardeners, we need to analyze the property of V. mandarinia venom. We, therefore, isolate the venomous PLs and identify their properties. In addition, some medicinal plants from traditional folklore, commonly used as an antidote for snake bites and bee or wasp stings in Japan and China (Kan, 1975), were studied. The eects of biscoclaurine alkaloids from the plants, such as cepharanthine contained in a root of Stephania cepharantha often used to remedy the in¯ammation and swelling of bites and stings (Abe et al., 1991a; Ishihara et al., 1995; Kosaka et al., 1996), were analyzed and characterized on the hornet PLs.
2. Materials and methods 2.1. Materials L-a-phosphatidylcholine-b-oleoil-g-palmitoil, palmitic acid, oleic acid, 4methanesulfonic acid, egg yolk lecithin and biscoclaurine alkaloids, such as cepharanthine, cycleanine, berbamine, isotetrandrine, tetrandorine were purchased from Wako Chemicals (Tokyo, Japan). Sephadex G-100, SP-Sepharose and Pharmalite were brought from Pharmacia (Upsara, Sweden). Bee venom PLA2, bovine pancreas PLA2 and Naja mossambica mossambica PLA2 were from Sigma (St. Louis, MO, USA). Standard protein for SDS electrophoresis was from
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Daiichi Yakuhin Chemicals (Tokyo, Japan). Marker proteins isoelectrofocussing was given from the Oriental Yeast (Tokyo, Japan).
for
2.2. Collection of the hornets Giant hornets (Vespa mandarinia ) were collected in Kanto plain in Japan. The venom sacs were removed from the venomous organs, and frozen quickly, then stored in liquid nitrogen until use. 2.3. Column chromatography for puri®cation of PLs All procedures were carried out at 0±48C. Homogenates containing venom and its sac (25% in 50 mM acetate buer, pH 5.2) were prepared using a Polytron homogenizer. The homogenate was centrifuged at 110,000 g for 60 min, and the supernatant was used as crude venom. The crude venom was chromatographed on a Sephadex G-100 column f 4.2 97 cm) equilibrated with 50 mM acetate buer, pH 5.2. Ten ml of each fraction were collected with an LKB Ultro Rac II fraction collector, monitored at 280 nm with a Pharmacia UV2 monitor. The enzyme active fractions were combined and concentrated by the Amicon concentration system using Dia¯ow membrane YM-5 under press of nitrogen gas (Abe et al., 1982). The concentrated active enzyme fraction was applied to SPSepharose column f 1.6 27 cm) chromatography. The column was fractionated 3 ml each by linear gradient using 50 mM acetate buer (150 ml), pH 5.6 and 50 mM acetate buer, pH 5.6 containing 0.8 M NaCl (150 ml). 2.4. Polyacrylamide gel electrophoreses (PAGE) for purity, molecular weight and isoelectric point determination of PLs SDS-PAGE was performed at 14% slab gel by the method of Laemmli (Laemmli, 1970). Twenty mg of standard protein was applied with 10±20 mg of samples. Acid PAGE was carried out by 10% slab gel at 35 mM b-alanine±acetate buer, pH 4.5 (Gabriel, 1971). Isoelectrofocussing PAGE was performed with 7.8% slab gel containing 10% Pharmalite (pH 7±10.5) (Vesterberg et al., 1977). The marker protein was migrated with samples. 2.5. Assay of enzyme activity for PL PL activity was assayed by 2% egg yolk lecithin containing 50 mg enzymes, 5 mM CaCl2 and 150 mM NaCl. Three ml of enzyme solution was incubated for 30 min at 378C in routine assay, or for 5, 15 and 30 min at 0±808C in the optimum temperature experiment. The reaction was stopped by addition of the same volume of ice cold water. Each reaction mixture was titrated by neutralization with 0.01 N NaOH, then calculated the reaction velocity. In the inhibition experiments, the enzyme reaction mixtures were contained 4.5 mM of cepharanthine, cycleanine, berbamine, isotetrandrine or tetrandrine. The inhibitory
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eect for the substrate of egg yolk lecithin was showed as percent based on without inhibitors. Analysis of temperature optimum for the substrate of L-aphosphatidylcholine-b-oleoil-g-parmitoil was performed for 60 min at 378C by the reaction mixture containing 250 mg substrate, 50 mM CaCl2, 115 mg PLB a or 230 mg PLB b, and 50 mM Tris±HCl buer, pH 7.5. The reaction was stopped by the addition of 1 ml chloroform. 2.6. Amino acid analyses for puri®ed enzymes Each 0.1 mg of lyophilized protein sample was hydrolyzed in 0.5 ml of 4 N methanesulfonic acid with 0.2% 3-(2-aminoethyl) indole at 1158C for 24 h. Analyses on amino acids were carried out by using an automated amino acid analyzer (Hitachi L-8500A). Each amino acid was detected by ninhydrin. 2.7. Enzyme kinetics for inhibitory properties Puri®ed enzymes were analyzed of the kinetical properties Km for egg yolk lecithin and Ki for biscoclaurine alkaloids. For analysis of active site speci®city of puri®ed PLB a and b, L-a-phosphatidylcholine-b-oleoil-g-parmitoil as substrate was used. One ml of the reaction mixture contained 0.1±0.5 mM L-aphosphatidylcholine-b-oleoil-g-parmitoil, 50 mM CaCl2, 100 mg PLB a or 50 mg PLB b, and 50 mM Tris±HCl buer, pH 7.5. The enzyme reaction was carried out for 0, 15 and 30 min at 378C. The reaction was stopped by the addition of 1 ml chloroform. The reaction velocity was measured for 0.1±0.5 mM of dierent substrate concentrations. The Km values were analyzed by Lineweaver Burk plot using the method of least squares. The Ki values of cepharanthine for PLB a and b, bee venom PLA2, bovine pancreas PLA2 and Naja mossambica mossambica PLA2 were analyzed 2.5±160 mg/ml of egg yolk lecithin as substrate by the same method as the Km value estimation. The three dierent cepharanthine concentrations of 6, 12.5 and 25 mM were performed, and also analyzed the nature of inhibition by Lineweaver Burk plot. 2.8. Gas chromatographic mass spectrometry for PL hydrolyzates Fatty acid hydrolyzates from L-a-phosphatidylcholine-b-oleoil-g-parmitoil which catalyzed by the puri®ed PLBs were extracted three times by 1 ml chloroform, then analyzed by a gas chromatographic mass spectrometer (HP5890II and 5971A, Hewlett Packard). The gas chromatography was equipped capillary chromatography of HP-INNOWax (cross linked polyethylene glycol, 30 m 0.25 mm 0.25 mm) and analyzed under temperature gradient from 1208C to 2508C. Both oleic acid and palmitic acid showed the retention times at 9.3 and 13.8 min, respectively.
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2.9. Other methods Protein concentration was determined by the method of Lowry et al. (1951). Bovine serum albumin was used for standard protein. 3. Results 3.1. Puri®cation of the hornet venomous PL Forty-four ml of the crude venom was applied to Sephadex G-100 column, equilibrated to 50 mM acetate buer, pH 5.2. The enzyme activity was fractionated a single peak at fraction no. 60±85. The active fraction was concentrated to 14 ml, then given to SP-Sepharose column (Fig. 1). The enzyme activity was separated into two peaks, a and b. Two molecular species of PLB were puri®ed by simpli®ed two step method. The puri®cation procedure and yield were shown in the Table 1. Two active fractions, a and b, concentrated by Dia¯ow membrane, were given a single protein band of enzyme by the slab gel of SDS-PAGE (Fig. 2) the acidic PAGE and the isoelectric focusing PAGE. 3.2. Physico-chemical properties of the puri®ed hornet venomous PLB The molecular size of the puri®ed a and b enzymes were estimated at 29.5 and 26.0 kDa, respectively, by SDS-PAGE (Fig. 2). Both enzymes were very labile for freeze-thawing, high pH and room temperature. The optimum temperatures of a and b enzymes were broad peaks at 40±608C for egg yolk lecithin, but for L-aphosphatidylcholine-b-oleoil-g-parmitoil at 40±558C (Fig. 3). The amino acid compositions of a and b enzymes were shown in Table 3. Both enzymes showed similar amino acid compositions, and high contents of aspartic acid, glycine,
Fig. 1. SP-Sepharose column chromatography of PL active fraction from Sephadex G-100 column chromatography. Experimental conditions are given in Section 2.
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Table 1 Puri®cation and yields of V. mandarinia PLs Column chromatography
Fractions
Total protein (mg)
Speci®c activity (unit)a
Yield (%)
Sephadex G-100 SP-Sepharose
Crude venom I Ia Ib
607.5 149.9 10.7 24.3
5.53 10.88 64.33 9.42
100.0 48.5 20.4 6.8
a
One unit is 1 ml of 0.01 N NaOH used for neutralization by 1 mg of enzyme proteins.
leucine, lysine and other aliphatic amino acids, such as alanine, valine and isoleucine. Furthermore, cystine was as commonly rich as other PLAs and PLBs (Scott and Sigler, 1994). a or b enzymes was peculiar high content of proline or threonine, respectively. The isoelectric points of a and b enzymes were pH 10.56 and 10.69, respectively. Both enzymes were very basic protein. 3.3. Kinetical properties of the PLB a and b The Km values of PLB a and b for egg yolk lecithin were 8.29 and 7.53 mg/ml, respectively (Table 2). Both enzymes hydrolyzed b and g residues of aphosphatidylcholine. The ratio of hydrolytic products were 63.8% of palmitic acid and 36.2% of oleic acid for PLB a and 20% and 80%, respectively, for PLB b. These results showed that PLB a catalyzed more A1 site than A2 site, but, PLB b catalyzed more A2 site than A1 site. In case of PLB a, the Km values for b-oleoil and g-palmitoil residues were 1.392 and 0.528 mM, respectively (Fig. 4). While the Km values of PLB b for both residues were 2.68 and 7.91 mM, respectively. The
Fig. 2. SDS-PAGE of puri®ed PLB a and b. a and b are PLB a and PLB b, respectively. Std is standard protein mixture. Experimental conditions are given in Section 2.
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Fig. 3. Temperature optima of PLB a and b. (A) Hydrolytic activities of PLB a (q) and PLB b (r) for egg yolk lecithin. (B) Hydrolysis of L-a-phosphatidylcholine-b-oleoil-g-palmitoil by PLB a and b. Palmitic acid by PLB a (r) and by PLB b (*). Oleic acid by PLB a (q) and by PLB b (w). Experimental conditions are given in Section 2.
Table 2 Inhibitory interaction of cepharanthine and anity with PLs of hornet (V. mandarinia ) venom, bee (A. mellifera ) venom, Naja mossambica mossambica and bovine pancreas
PLB a PLB b Bee PLA2 Naja mossambica mossambica PLA2 Bovine pancreas PLA2
Km for egg yolk lecithin (g/ml) Ki (M)
Nature of inhibition
8.29 10ÿ3 7.53 10ÿ3 76.18 10ÿ3 62.53 10ÿ3 9.31 10ÿ3
Competitive Uncompetitive Noncompetitive Noncompetitive Noncompetitive
36.04 10ÿ9 40.81 10ÿ9 1.24 10ÿ9 1.82 10ÿ9 31.05 10ÿ9
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Fig. 4. Position anity of PLB a and b for L-a-phosphatidylcholine-b-oleoil-g-palmitoil. (A) Palmitoil residue (*), y = 681.5x + 1.698 (r = 0.978). Oleoil residue (w), y = 874.2x + 0.8263 (r = 0.979). (B) Palmitoil residue (*), y = 695.6x + 0.1157 (r = 0.992). Oleoil resude (w), y = 562.3x + 0.2766 (r = 0.988). Experimental conditions are given in Section 2.
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substrate anity was higher PLB a than b, but in both enzymes, the active sites of A1 and A2 were characterized by an opposite anity, that is, a and b enzymes had higher anity to A1 and A2 sites, respectively. Both enzymes had dierent enzymatic characterization. 3.4. Inhibition of PL activities by biscoclaurine alkaloids The inhibitory eects of biscoclaurine alkaloids on the puri®ed PLB a and b were analyzed as shown in Fig. 5. The PLB a activity was inhibited 8±25% in order from berbamine, to cycleanine, to tetrandrine and to isotetrandrine (Fig. 6). The PLB b activity was similarly inhibited 7±30% in order from berbamine, to cycleanine and to isotetrandrine, but activated by tetrandrine. On the other hand, both enzymes were inhibited more than 70% by cepharanthine. The results
Fig. 5. Structure of biscoclaurine alkaloids.
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suggest that it seems to have a reducing eect of cepharanthine for edema and pain from hornet stings. 3.5. Kinetical analyses of cepharanthine inhibition for several PLs The inhibitory eect of cepharanthine to giant hornet venomous PLB a and b was analyzed and listed in Table 2. The PLB a activity against egg yolk lecithin was competitively inhibited. The Km values of giant hornet PLB a and b, and bovine pancreas PLA2 for egg yolk lecithin were characterized by higher anity. The other Km values of Naja mossambica mossambica and bee venom PLA2s were about 10 times higher than the former enzymes. While, the Ki values of bee venom and Naja mossambica mossambica PLA2s for cepharanthine were lower than that of hornet PLBs and bovine pancreas PLA2. Hornet PLB a and b were inhibited competitively and uncompetitively, respectively, by cepharanthine. But, PLA2s of bee venom, Naja mossambica mossambica and bovine pancreas were non competitive inhibition. These results suggest that cepharanthine inhibits many PL activities. 4. Discussion The role of the hornet venomous PLs is an oensive as well as defensive armament in the weaponry of hornets. The venom is not only used as toxins for preyed insects, but also digests their cell wall components of diacylphospholipid, such as phosphatidylcholine, phophatidylserine and phosphatidylethanolamine, to fatty acids and phospholipids by containing PLs. At this moment, PLBs are more universally digestive enzymes than PLA1 or A2 by itself, because both A1 and A2
Fig. 6. Inhibition of PLB a (q) and b (Q) by 4.5 mmol biscoclaurine alkaloids. Cep: cepharanthine, Cyc: cycleanine; Ber: berbamine; Iso: isotetrandrine; Tet: tetrandrine. The enzymatic inhibition was calculated as percent activity against without inhibitors. Experimental conditions are given in Section 2.
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sites are hydrolyzed by one enzyme. It seems to be a kind of enzymatic adaptation of the carnivorous insects, Vespa, against vegetarian insects, Apis, which contain only PLA2 in the venom. The counterpart liquid is amino acid rich lymph of preyed insects and soluble fatty acids digested with venomous enzymes into meatball minced preyed insects. These are eaten by adult hornets with a narrow esophagus through the narrow part of petiolus. The remaining solid meatball is carried to the larva living in the nest. The reasons why the universally digestive enzymes are needed lie in the ¯ight energy metabolism of the hornet. The ¯ight energy of insects commonly relies on fatty acid oxidation, especially for heavy body weight and long distance ¯ights (Beenakkers, 1965) because of the prevention of the production of fatigue substances which most likely is lactic acid. Of course, a nature of a light and high calorie fat diet are an advantage for ¯ight. It is known that the fatty acid oxidation in the hornets is promoted by their larval saliva (Abe et al., 1991b; 1997). The social insects have the habit of exchanging their foods between adults and larva, named trophallaxis (Wilson, 1971). In comparison to the physico-chemical properties of PLB a and b from V. mandarinia, the amino acid compositions were very similar between both enzymes, except that proline and leucine are rich in PLB a and alanine and histidine are rich in PLB b. However, both between PLB I of V. xanthoptera and PLB a of V. mandarinia and between PLB II and PLB b had much more similar amino acid compositions than others (Table 3). In spite of the similarity of amino acid composition, isoelectric points of both PLBs from V. mandarinia and V. xanthoptera were markedly dierent at 10.6±10.7 and at 6.4±6.5, respectively (Takasaki and Fukumoto, 1989). The isoelectric points of other PLs and proteinous toxins from many species of hornets and wasps are of signi®cantly basic properties (Abe et al., 1982; Ho and Ko, 1988). On the other hand, the molecular sizes of PLs in hornets and wasps are about 26±37 kDa (Table 3), and mainly distributed around 30 kDa (Table 3). Mandaratoxin and Antigen 5 are signi®cantly similar amino acid compositions to each other, but have dierent molecular weights (Abe et al., 1982; King et al., 1984). In the series of those proteins having similar amino acid compositions, aspartic acid, serine, glycine, cysteine, isoleucine, tyrosine, lysine and arginine are unchangable common components, except hornetin which has no cysteine content. It could be suggested that those proteins would be dierentiated from the common original protein like the lethal protein of V. basalis. It is well known that many PLBs from hornets and wasps are unstable for higher pH, freeze-thawing and room temperature (Ho and Ko, 1988; Takasaki and Fukumoto, 1989). The puri®ed PLB a and b were also very labile during and after puri®cation. Both enzymes had not only the enzyme activity, but also hemolytic activity and cardiotoxicity (unpublished data). Other hornet and wasp PLs show multiple functional properties of hemolysis (Ho and Ko, 1986, 1988; DeOliveira and Palma, 1998), presynaptic neurotoxicity (Ho and Ko, 1986), lethal toxicity (Ho and Ko, 1988), cardiotoxicity (Ho and Ko, 1988), edema inducing activity (Ho et al., 1993), allergen (King et al., 1984) and so on (Table 3). The multiple toxic functions of PLB seem to have stronger eects than phospholipase
9.5 5.4 6.9 5.9 8.0 8.9 5.6 2.0 5.8 1.3 6.6 8.8 4.9 4.7 10.0 2.4 3.4 nd 29.5 10.6 B + Edema cardiotoxicity
PLB a
V. mandarinia
9.9 7.6 6.1 6.3 5.4 8.3 6.9 1.9 6.3 1.6 6.1 7.6 5.3 3.9 10.3 3.6 2.8 0.1 26.0 10.7 B +++ Edema
10.1 5.4 5.1 10.8 5.2 9.4 6.9 2.7 5.8 1.8 5.9 5.2 4.8 2.1 10.2 3.1 3.1 2.5 21.0 9.1 nd ÿ Neurotoxicity
PLB b MDTb 11.0 5.4 7.0 9.5 5.0 8.3 4.8 3.7 5.0 1.6 6.2 7.7 5.0 4.8 9.3 2.4 3.2 nd 30.9 6.4 B nd
10.4 7.7 5.7 7.9 4.7 8.3 7.4 2.5 6.3 0.6 6.3 7.1 4.9 3.9 9.6 4.0 2.7 nd 30.9 6.5 B nd
PLB I PLB II
V. xanthoptera
9.9 6.7 5.6 7.3 5.0 7.6 6.4 3.7 6.8 2.7 6.3 7.3 5.0 3.7 9.3 3.5 3.0 0.3 31.8 nd A1 +++ Edema lethaltoxicity cardiotoxicity
LTc
V. basalis
b
Bold number shows a marked dierence from other species, nd: no detectable. na: no analysis. MDT: mandaratoxin. c LT: lethal toxin. d HO: hornetin. e Ant5: antigen 5. f Detected by SDS-PAGE.
a
Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Try Molecular weight (kDa)f Isoelectric point (pH) Type of enzyme Hemolysis Other properties identi®ed
Amino acids
Table 3 Amino acid compositions and physico-chemical properties of PLs from hornets and waspa
11.3 5.5 3.0 8.3 5.3 8.6 7.0 0 7.6 2.6 6.5 7.9 4.2 4.0 10.9 4.3 2.9 0 32.0 10.2 na +++ Edema neurotoxicity
HOd
V. ¯avitarsus
11.7 5.2 5.6 11.8 3.9 9.4 6.8 4.3 5.9 1.6 5.4 5.6 4.9 2.1 9.9 3.2 2.6 nd 27.0 nd nd nd Allergen
Ant5e
P. annularis
11.1 5.4 6.7 6.7 3.2 8.0 8.3 4.1 7.3 0.6 5.4 7.6 4.8 4.8 9.6 3.2 3.2 nd 37.0 nd B nd
PLB
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A2. Both puri®ed enzymes should be designated with a name such as Kumantoxin a and b to be associated with the traditional Japanese hornet name in the same way as many multifunctional enzymes simultaneously have particular toxin names. The puri®ed PLB a and b showed a catalytic or anity inclination on b and g residues of phosphatidylcholine. The PLB a had the higher anity for g position, but the PLB b had less anity for g position in our experiments. While, the amino acid compositions of PLBs from V. xanthoptera are compared to those from V. mandarinia, PLB a and b are corresponded to PLB I and PLB II, respectively (Table 3), despite a marked dierence between both kinetic properties, PLB I and PLB II were likely PLA1 type, but those of PLB a and b were strictly the types of PLB. We need more precise experiments to show whether the dierences of fatty acids residues in the b and g position in¯uence the enzymatic activity or not. The Indian plant, Aristolochia indica, is used for treating poisonous snake bites (Tsai et al., 1975). It has been found that aristolochic acid contained in the plant inhibits the activity of PLA2 from Viperid and Crotalid, and of other PLA2 from poisonous snakes (Vishwanath et al., 1987a; Vishwanath and Gowda, 1987b). The other alkaloids and plant isolates such as wedelolactone (from Eclipta prostrata ) (Melo et al., 1995), stigma sterol (from E.prostreta ), b-sitosterol (from E. prostrata ) and cabenegrin A-I and A-II (Cabeca-de negro) (Nakagawa et al., 1982) aect the inhibitory of PLA2. The Ki values of those anti PLA2 compounds were a similar range of cepharanthine from Stephania cepharantha (Table 2). Cepharanthine markedly inhibited the activities of PLB a and b from V. mandarinia, PLA2s from bee venom, Naja mossambica mossambica and bovine pancreas. The dierence of inhibitory actions between competitive or uncompetitive for hornet PLBs and noncompetitive for other PLA2s would suggest the characteristic dierences between PLB and PLA2. While the structurally similar compounds of berbamine, cycleanine, tetrandrine and isotetrandrine showed very low inhibitory activity. These results suggest that an analysis of the structural and catalitic correlation would provide useful knowledge for the enzymatic control of PLB. Acknowledgements We would like to thank Mr. Masao Chijimatsu of our institute for amino acid analysis. References Abe, T., Kawai, N., Niwa, A., 1982. Puri®cation and properties of a presynaptically active neurotoxin, mandaratoxin, from hornet (Vespa mandarinia ). Biochemistry 21, 1693±1697. Abe, T., Inamura, S., Akasu, M., 1991a. Eect of cepharanthin on the lethality and cardiovascular disorder by Mamushi, Agkistrodon halys blomhoi, snake venom. Folia Pharmacol (Japan) 98, 327± 336.
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Abe, T., Tanaka, Y., Miyazaki, H., Kawasaki, Y., 1991b. Comparative study of the composition of hornet larval saliva, its eect on behavior and role of trophallaxis. Comp. Biochem. Physiol. 99C, 79±84. Abe, T., Inamori, M., Iida, K., Tamura, M., Takiguchi, Y., Yasuda, K., 1997. The activation of fatty acid metabolism by Vespa amino acid mixture (VAAM) and related nutrients during endurance exercise in mice. Adv. Exerc. Sports Physiol. 3, 35±44. Beenakkers, A.M.T., 1965. Transport of fatty acids in Locusta migratoria during sustained ¯ight. J. Insect Physiol. 11, 879±888. De Oliveira, M.R., Palma, M.S., 1998. Polybiotoxins: a group of phospholipases A2 from the venom of the neotropical social wasp Paulistinha (Polybia paulista ). Gabriel, O., 1971. Analytical disc gel electrophoresis. Method Enzymol. 22, 565±578. Habermann, E., Neumann, W.P., 1957. Reinigung der phospholipase A des bienengiftes. Biochem. Z. 328, 465±473. Ho, C.L., Ko, J.L., 1986. Hornetin: the lethal protein of the hornet (Vespa ¯avitarsus ). FEBS Lett. 209, 18±22. Ho, C.L., Ko, J.L., 1988. Puri®cation and characterization of a lethal protein with phospholipases A1 activity from the hornet (Vespa basalis ) venom. Biochem. Biophys. Act. 963, 414±422. Ho, C.L., Hwang, L.L., Chen, C.T., 1993. Edema-inducing activity of a lethal protein with phospholipase A1 activity isolated from the black-bellied hornet (Vespa basalis ) venom. Toxicon 31, 605±613. Ishihara, M., Abe, M., Urashima, R., Sano, Y., Mihara, M., 1995. Clinical study of 39 cases of ``Mamushi'' viper bite over the past 4 years. Jpn. J. Dermatol. 105, 1431±1437. Kan, W.S., 1975. Pharmaceutical Botany. National Research Institute of Chinese Medicine, Taipei, Republic of China, pp. 280±281. King, T.P., Kochoumian, L., Joslyn, A., 1984. Wasp venom proteins : phospholipase A1 and B. Arch. Biochem. Biophys. 230, 1±12. Kini, R.M. (Ed.), 1997. Venom Phospholipase A2 Enzyme, Structure, Function and Mechanism. Wiley, Chichester, UK. Kosaka, M., Goto, K., Ota, Y., Hirakawa, M., Ogino, K., 1996. A study of antivenin therapy and cepharanthin therapy for mamusni bite. ICU and CCU 20, 243±249. Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265±275. Melo, P.A., do Nascimeto, M.C., Mors, W.B., Suarez-Kurtz, G., 1995. Inhibition of myotoxic and hemorrhagic activities of crotalid venoms by Eclipta prostrata (Asteraceae) extracts and constituents. Toxicon 32, 595±603. Nakagawa, M., Nakanishi, K., Darko, L.L., Vick, J.A., 1982. Structures of cabenegrins A-I and A-II, potent anti-snake venoms. Tetrahedron Lett. 3855±3858. Scott, D.L., Sigler, P.B., 1994. Structure and catalytic mechanism of secretory phospholipase A2. Adv. Protein Chem. 45, 53±88. Takasaki, C., Fukumoto, M., 1989. Phospholipases B from Japanese yellow hornet (Vespa xanthoptera ) venom. Toxicon 27, 449±458. Tsai, L.H., Lie, H.J., Yang, C.P., Chang, C., 1975. Inactivation of formosan snake venoms in vitro by the crude extract of Aristochia radix. J. Formosan Med. Assoc. 74, 352±360. Vesterberg, O., Hausen, L., Sjosten, A., 1977. Staining of proteins after isoelectric focusing in gels by new procedures. Biochem. Biophys. Act. 491, 160±166. Vishwanath, B.S., Kini, R.M., Gowda, T.V., 1987a. Characterization of three edema-inducing phospholipase A2 enzymes from Habu (Trimeresurus ¯avoviridis ) Venom and their interaction with the alkaloid aristolochic acid. Toxicon 25, 501±515. Vishwanath, B.S., Gowda, T.V., 1987b. Interaction of aristolochic acid with Vipera russelli phospholipase A2: its eect on enzymatic and pathological activities. Toxicon 25, 929±937. Wilson, E.O., 1971. The Insect Societies, Communication: Recognition, Food Exchange and Grooming. Berknap Press, Harvard, pp. 272±295.
Giant hornet (Vespa mandarinia ) venomous phospholipases The puri®cation, characterization and inhibitory properties by biscoclaurine alkaloids Takashi Abe a,*, Masato Sugita a, Tsuyoshi Fujikura a, Jiro Hiyoshi a, Michinori Akasu b a
Hornet Research Group, The Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan b Research laboratory, Kaken Shoyaku Pharmaceutical Co.3-37-10, Shimorenjaku, Mitaka, Tokyo 181-0013, Japan Received 6 July 1999; accepted 8 February 2000
Abstract Two species of giant hornet phospholipase B (PLB), a and b, were puri®ed from the venom of Vespa mandarinia. The puri®cation procedure was simpli®ed by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB a and b were 29.5 and 26.0 kDa, respectively. The isoelectric point of a and b enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40±608C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The Km values of a and b enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic speci®city for L-a-phosphatidylcholine-b-oleoil-g-palmitoil, the Km values of a enzyme for g-palmitoil and b-oleoil residues were 0.528 and 1.392 mM, respectively. While the Km values of b enzyme for g-palmitoil and b-oleoil residues were 7.91 and 2.68 mM, respectively. Both a and b enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of a enzyme was competitively inhibited, but b enzyme was uncompetitive.
* Corresponding author. Tel.: +81-48-467-9527; fax: +81-48-462-1552. E-mail address: [email protected] (T. Abe). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 1 0 9 - 4
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Cepharanthine also inhibited noncompetitively PLA2s of bovine pancreas, bee venom and Naja mossambica mossambica. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phospholipase B; Biscoclaurine alkaloids; Cepharanthine; Hornet; Venom; Vespa mandarinia
1. Introduction Venomous PLS found in poisonous animals, especially PLA2s in snake venoms, are widely studied in molecular biology and pharmacology (Kini, 1997). While, in hymenoptera venoms, wasp and hornet venomous PLs are characterized as PLA1, PLA2 and PLB, despite that of bee, Apis mellifera, venom is PLA2 (Habermann and Neumann, 1957). In hornet species of Vespa xanthoptera (Takasaki and Fukumoto, 1989) and V. basalis (Ho and Ko, 1988), only the PLs were puri®ed and characterized. Therefore, we have not yet reached the knowledge of the common properties of the hornet venomous PLs which also have multiple functions, and is one of the most toxic components in the venoms. On the other hand, we still have serious hornet stung injuries, especially by giant hornets, V. mandarinia, in Japan. Over 40 of those injured have been killed by wasp stings each year. However, there have not been better therapy to cure the sting injuries. Especially for the demand to save lives of bee keepers, lumbermen, farmers and gardeners, we need to analyze the property of V. mandarinia venom. We, therefore, isolate the venomous PLs and identify their properties. In addition, some medicinal plants from traditional folklore, commonly used as an antidote for snake bites and bee or wasp stings in Japan and China (Kan, 1975), were studied. The eects of biscoclaurine alkaloids from the plants, such as cepharanthine contained in a root of Stephania cepharantha often used to remedy the in¯ammation and swelling of bites and stings (Abe et al., 1991a; Ishihara et al., 1995; Kosaka et al., 1996), were analyzed and characterized on the hornet PLs.
2. Materials and methods 2.1. Materials L-a-phosphatidylcholine-b-oleoil-g-palmitoil, palmitic acid, oleic acid, 4methanesulfonic acid, egg yolk lecithin and biscoclaurine alkaloids, such as cepharanthine, cycleanine, berbamine, isotetrandrine, tetrandorine were purchased from Wako Chemicals (Tokyo, Japan). Sephadex G-100, SP-Sepharose and Pharmalite were brought from Pharmacia (Upsara, Sweden). Bee venom PLA2, bovine pancreas PLA2 and Naja mossambica mossambica PLA2 were from Sigma (St. Louis, MO, USA). Standard protein for SDS electrophoresis was from
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Daiichi Yakuhin Chemicals (Tokyo, Japan). Marker proteins isoelectrofocussing was given from the Oriental Yeast (Tokyo, Japan).
for
2.2. Collection of the hornets Giant hornets (Vespa mandarinia ) were collected in Kanto plain in Japan. The venom sacs were removed from the venomous organs, and frozen quickly, then stored in liquid nitrogen until use. 2.3. Column chromatography for puri®cation of PLs All procedures were carried out at 0±48C. Homogenates containing venom and its sac (25% in 50 mM acetate buer, pH 5.2) were prepared using a Polytron homogenizer. The homogenate was centrifuged at 110,000 g for 60 min, and the supernatant was used as crude venom. The crude venom was chromatographed on a Sephadex G-100 column f 4.2 97 cm) equilibrated with 50 mM acetate buer, pH 5.2. Ten ml of each fraction were collected with an LKB Ultro Rac II fraction collector, monitored at 280 nm with a Pharmacia UV2 monitor. The enzyme active fractions were combined and concentrated by the Amicon concentration system using Dia¯ow membrane YM-5 under press of nitrogen gas (Abe et al., 1982). The concentrated active enzyme fraction was applied to SPSepharose column f 1.6 27 cm) chromatography. The column was fractionated 3 ml each by linear gradient using 50 mM acetate buer (150 ml), pH 5.6 and 50 mM acetate buer, pH 5.6 containing 0.8 M NaCl (150 ml). 2.4. Polyacrylamide gel electrophoreses (PAGE) for purity, molecular weight and isoelectric point determination of PLs SDS-PAGE was performed at 14% slab gel by the method of Laemmli (Laemmli, 1970). Twenty mg of standard protein was applied with 10±20 mg of samples. Acid PAGE was carried out by 10% slab gel at 35 mM b-alanine±acetate buer, pH 4.5 (Gabriel, 1971). Isoelectrofocussing PAGE was performed with 7.8% slab gel containing 10% Pharmalite (pH 7±10.5) (Vesterberg et al., 1977). The marker protein was migrated with samples. 2.5. Assay of enzyme activity for PL PL activity was assayed by 2% egg yolk lecithin containing 50 mg enzymes, 5 mM CaCl2 and 150 mM NaCl. Three ml of enzyme solution was incubated for 30 min at 378C in routine assay, or for 5, 15 and 30 min at 0±808C in the optimum temperature experiment. The reaction was stopped by addition of the same volume of ice cold water. Each reaction mixture was titrated by neutralization with 0.01 N NaOH, then calculated the reaction velocity. In the inhibition experiments, the enzyme reaction mixtures were contained 4.5 mM of cepharanthine, cycleanine, berbamine, isotetrandrine or tetrandrine. The inhibitory
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eect for the substrate of egg yolk lecithin was showed as percent based on without inhibitors. Analysis of temperature optimum for the substrate of L-aphosphatidylcholine-b-oleoil-g-parmitoil was performed for 60 min at 378C by the reaction mixture containing 250 mg substrate, 50 mM CaCl2, 115 mg PLB a or 230 mg PLB b, and 50 mM Tris±HCl buer, pH 7.5. The reaction was stopped by the addition of 1 ml chloroform. 2.6. Amino acid analyses for puri®ed enzymes Each 0.1 mg of lyophilized protein sample was hydrolyzed in 0.5 ml of 4 N methanesulfonic acid with 0.2% 3-(2-aminoethyl) indole at 1158C for 24 h. Analyses on amino acids were carried out by using an automated amino acid analyzer (Hitachi L-8500A). Each amino acid was detected by ninhydrin. 2.7. Enzyme kinetics for inhibitory properties Puri®ed enzymes were analyzed of the kinetical properties Km for egg yolk lecithin and Ki for biscoclaurine alkaloids. For analysis of active site speci®city of puri®ed PLB a and b, L-a-phosphatidylcholine-b-oleoil-g-parmitoil as substrate was used. One ml of the reaction mixture contained 0.1±0.5 mM L-aphosphatidylcholine-b-oleoil-g-parmitoil, 50 mM CaCl2, 100 mg PLB a or 50 mg PLB b, and 50 mM Tris±HCl buer, pH 7.5. The enzyme reaction was carried out for 0, 15 and 30 min at 378C. The reaction was stopped by the addition of 1 ml chloroform. The reaction velocity was measured for 0.1±0.5 mM of dierent substrate concentrations. The Km values were analyzed by Lineweaver Burk plot using the method of least squares. The Ki values of cepharanthine for PLB a and b, bee venom PLA2, bovine pancreas PLA2 and Naja mossambica mossambica PLA2 were analyzed 2.5±160 mg/ml of egg yolk lecithin as substrate by the same method as the Km value estimation. The three dierent cepharanthine concentrations of 6, 12.5 and 25 mM were performed, and also analyzed the nature of inhibition by Lineweaver Burk plot. 2.8. Gas chromatographic mass spectrometry for PL hydrolyzates Fatty acid hydrolyzates from L-a-phosphatidylcholine-b-oleoil-g-parmitoil which catalyzed by the puri®ed PLBs were extracted three times by 1 ml chloroform, then analyzed by a gas chromatographic mass spectrometer (HP5890II and 5971A, Hewlett Packard). The gas chromatography was equipped capillary chromatography of HP-INNOWax (cross linked polyethylene glycol, 30 m 0.25 mm 0.25 mm) and analyzed under temperature gradient from 1208C to 2508C. Both oleic acid and palmitic acid showed the retention times at 9.3 and 13.8 min, respectively.
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2.9. Other methods Protein concentration was determined by the method of Lowry et al. (1951). Bovine serum albumin was used for standard protein. 3. Results 3.1. Puri®cation of the hornet venomous PL Forty-four ml of the crude venom was applied to Sephadex G-100 column, equilibrated to 50 mM acetate buer, pH 5.2. The enzyme activity was fractionated a single peak at fraction no. 60±85. The active fraction was concentrated to 14 ml, then given to SP-Sepharose column (Fig. 1). The enzyme activity was separated into two peaks, a and b. Two molecular species of PLB were puri®ed by simpli®ed two step method. The puri®cation procedure and yield were shown in the Table 1. Two active fractions, a and b, concentrated by Dia¯ow membrane, were given a single protein band of enzyme by the slab gel of SDS-PAGE (Fig. 2) the acidic PAGE and the isoelectric focusing PAGE. 3.2. Physico-chemical properties of the puri®ed hornet venomous PLB The molecular size of the puri®ed a and b enzymes were estimated at 29.5 and 26.0 kDa, respectively, by SDS-PAGE (Fig. 2). Both enzymes were very labile for freeze-thawing, high pH and room temperature. The optimum temperatures of a and b enzymes were broad peaks at 40±608C for egg yolk lecithin, but for L-aphosphatidylcholine-b-oleoil-g-parmitoil at 40±558C (Fig. 3). The amino acid compositions of a and b enzymes were shown in Table 3. Both enzymes showed similar amino acid compositions, and high contents of aspartic acid, glycine,
Fig. 1. SP-Sepharose column chromatography of PL active fraction from Sephadex G-100 column chromatography. Experimental conditions are given in Section 2.
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Table 1 Puri®cation and yields of V. mandarinia PLs Column chromatography
Fractions
Total protein (mg)
Speci®c activity (unit)a
Yield (%)
Sephadex G-100 SP-Sepharose
Crude venom I Ia Ib
607.5 149.9 10.7 24.3
5.53 10.88 64.33 9.42
100.0 48.5 20.4 6.8
a
One unit is 1 ml of 0.01 N NaOH used for neutralization by 1 mg of enzyme proteins.
leucine, lysine and other aliphatic amino acids, such as alanine, valine and isoleucine. Furthermore, cystine was as commonly rich as other PLAs and PLBs (Scott and Sigler, 1994). a or b enzymes was peculiar high content of proline or threonine, respectively. The isoelectric points of a and b enzymes were pH 10.56 and 10.69, respectively. Both enzymes were very basic protein. 3.3. Kinetical properties of the PLB a and b The Km values of PLB a and b for egg yolk lecithin were 8.29 and 7.53 mg/ml, respectively (Table 2). Both enzymes hydrolyzed b and g residues of aphosphatidylcholine. The ratio of hydrolytic products were 63.8% of palmitic acid and 36.2% of oleic acid for PLB a and 20% and 80%, respectively, for PLB b. These results showed that PLB a catalyzed more A1 site than A2 site, but, PLB b catalyzed more A2 site than A1 site. In case of PLB a, the Km values for b-oleoil and g-palmitoil residues were 1.392 and 0.528 mM, respectively (Fig. 4). While the Km values of PLB b for both residues were 2.68 and 7.91 mM, respectively. The
Fig. 2. SDS-PAGE of puri®ed PLB a and b. a and b are PLB a and PLB b, respectively. Std is standard protein mixture. Experimental conditions are given in Section 2.
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Fig. 3. Temperature optima of PLB a and b. (A) Hydrolytic activities of PLB a (q) and PLB b (r) for egg yolk lecithin. (B) Hydrolysis of L-a-phosphatidylcholine-b-oleoil-g-palmitoil by PLB a and b. Palmitic acid by PLB a (r) and by PLB b (*). Oleic acid by PLB a (q) and by PLB b (w). Experimental conditions are given in Section 2.
Table 2 Inhibitory interaction of cepharanthine and anity with PLs of hornet (V. mandarinia ) venom, bee (A. mellifera ) venom, Naja mossambica mossambica and bovine pancreas
PLB a PLB b Bee PLA2 Naja mossambica mossambica PLA2 Bovine pancreas PLA2
Km for egg yolk lecithin (g/ml) Ki (M)
Nature of inhibition
8.29 10ÿ3 7.53 10ÿ3 76.18 10ÿ3 62.53 10ÿ3 9.31 10ÿ3
Competitive Uncompetitive Noncompetitive Noncompetitive Noncompetitive
36.04 10ÿ9 40.81 10ÿ9 1.24 10ÿ9 1.82 10ÿ9 31.05 10ÿ9
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Fig. 4. Position anity of PLB a and b for L-a-phosphatidylcholine-b-oleoil-g-palmitoil. (A) Palmitoil residue (*), y = 681.5x + 1.698 (r = 0.978). Oleoil residue (w), y = 874.2x + 0.8263 (r = 0.979). (B) Palmitoil residue (*), y = 695.6x + 0.1157 (r = 0.992). Oleoil resude (w), y = 562.3x + 0.2766 (r = 0.988). Experimental conditions are given in Section 2.
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substrate anity was higher PLB a than b, but in both enzymes, the active sites of A1 and A2 were characterized by an opposite anity, that is, a and b enzymes had higher anity to A1 and A2 sites, respectively. Both enzymes had dierent enzymatic characterization. 3.4. Inhibition of PL activities by biscoclaurine alkaloids The inhibitory eects of biscoclaurine alkaloids on the puri®ed PLB a and b were analyzed as shown in Fig. 5. The PLB a activity was inhibited 8±25% in order from berbamine, to cycleanine, to tetrandrine and to isotetrandrine (Fig. 6). The PLB b activity was similarly inhibited 7±30% in order from berbamine, to cycleanine and to isotetrandrine, but activated by tetrandrine. On the other hand, both enzymes were inhibited more than 70% by cepharanthine. The results
Fig. 5. Structure of biscoclaurine alkaloids.
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suggest that it seems to have a reducing eect of cepharanthine for edema and pain from hornet stings. 3.5. Kinetical analyses of cepharanthine inhibition for several PLs The inhibitory eect of cepharanthine to giant hornet venomous PLB a and b was analyzed and listed in Table 2. The PLB a activity against egg yolk lecithin was competitively inhibited. The Km values of giant hornet PLB a and b, and bovine pancreas PLA2 for egg yolk lecithin were characterized by higher anity. The other Km values of Naja mossambica mossambica and bee venom PLA2s were about 10 times higher than the former enzymes. While, the Ki values of bee venom and Naja mossambica mossambica PLA2s for cepharanthine were lower than that of hornet PLBs and bovine pancreas PLA2. Hornet PLB a and b were inhibited competitively and uncompetitively, respectively, by cepharanthine. But, PLA2s of bee venom, Naja mossambica mossambica and bovine pancreas were non competitive inhibition. These results suggest that cepharanthine inhibits many PL activities. 4. Discussion The role of the hornet venomous PLs is an oensive as well as defensive armament in the weaponry of hornets. The venom is not only used as toxins for preyed insects, but also digests their cell wall components of diacylphospholipid, such as phosphatidylcholine, phophatidylserine and phosphatidylethanolamine, to fatty acids and phospholipids by containing PLs. At this moment, PLBs are more universally digestive enzymes than PLA1 or A2 by itself, because both A1 and A2
Fig. 6. Inhibition of PLB a (q) and b (Q) by 4.5 mmol biscoclaurine alkaloids. Cep: cepharanthine, Cyc: cycleanine; Ber: berbamine; Iso: isotetrandrine; Tet: tetrandrine. The enzymatic inhibition was calculated as percent activity against without inhibitors. Experimental conditions are given in Section 2.
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sites are hydrolyzed by one enzyme. It seems to be a kind of enzymatic adaptation of the carnivorous insects, Vespa, against vegetarian insects, Apis, which contain only PLA2 in the venom. The counterpart liquid is amino acid rich lymph of preyed insects and soluble fatty acids digested with venomous enzymes into meatball minced preyed insects. These are eaten by adult hornets with a narrow esophagus through the narrow part of petiolus. The remaining solid meatball is carried to the larva living in the nest. The reasons why the universally digestive enzymes are needed lie in the ¯ight energy metabolism of the hornet. The ¯ight energy of insects commonly relies on fatty acid oxidation, especially for heavy body weight and long distance ¯ights (Beenakkers, 1965) because of the prevention of the production of fatigue substances which most likely is lactic acid. Of course, a nature of a light and high calorie fat diet are an advantage for ¯ight. It is known that the fatty acid oxidation in the hornets is promoted by their larval saliva (Abe et al., 1991b; 1997). The social insects have the habit of exchanging their foods between adults and larva, named trophallaxis (Wilson, 1971). In comparison to the physico-chemical properties of PLB a and b from V. mandarinia, the amino acid compositions were very similar between both enzymes, except that proline and leucine are rich in PLB a and alanine and histidine are rich in PLB b. However, both between PLB I of V. xanthoptera and PLB a of V. mandarinia and between PLB II and PLB b had much more similar amino acid compositions than others (Table 3). In spite of the similarity of amino acid composition, isoelectric points of both PLBs from V. mandarinia and V. xanthoptera were markedly dierent at 10.6±10.7 and at 6.4±6.5, respectively (Takasaki and Fukumoto, 1989). The isoelectric points of other PLs and proteinous toxins from many species of hornets and wasps are of signi®cantly basic properties (Abe et al., 1982; Ho and Ko, 1988). On the other hand, the molecular sizes of PLs in hornets and wasps are about 26±37 kDa (Table 3), and mainly distributed around 30 kDa (Table 3). Mandaratoxin and Antigen 5 are signi®cantly similar amino acid compositions to each other, but have dierent molecular weights (Abe et al., 1982; King et al., 1984). In the series of those proteins having similar amino acid compositions, aspartic acid, serine, glycine, cysteine, isoleucine, tyrosine, lysine and arginine are unchangable common components, except hornetin which has no cysteine content. It could be suggested that those proteins would be dierentiated from the common original protein like the lethal protein of V. basalis. It is well known that many PLBs from hornets and wasps are unstable for higher pH, freeze-thawing and room temperature (Ho and Ko, 1988; Takasaki and Fukumoto, 1989). The puri®ed PLB a and b were also very labile during and after puri®cation. Both enzymes had not only the enzyme activity, but also hemolytic activity and cardiotoxicity (unpublished data). Other hornet and wasp PLs show multiple functional properties of hemolysis (Ho and Ko, 1986, 1988; DeOliveira and Palma, 1998), presynaptic neurotoxicity (Ho and Ko, 1986), lethal toxicity (Ho and Ko, 1988), cardiotoxicity (Ho and Ko, 1988), edema inducing activity (Ho et al., 1993), allergen (King et al., 1984) and so on (Table 3). The multiple toxic functions of PLB seem to have stronger eects than phospholipase
9.5 5.4 6.9 5.9 8.0 8.9 5.6 2.0 5.8 1.3 6.6 8.8 4.9 4.7 10.0 2.4 3.4 nd 29.5 10.6 B + Edema cardiotoxicity
PLB a
V. mandarinia
9.9 7.6 6.1 6.3 5.4 8.3 6.9 1.9 6.3 1.6 6.1 7.6 5.3 3.9 10.3 3.6 2.8 0.1 26.0 10.7 B +++ Edema
10.1 5.4 5.1 10.8 5.2 9.4 6.9 2.7 5.8 1.8 5.9 5.2 4.8 2.1 10.2 3.1 3.1 2.5 21.0 9.1 nd ÿ Neurotoxicity
PLB b MDTb 11.0 5.4 7.0 9.5 5.0 8.3 4.8 3.7 5.0 1.6 6.2 7.7 5.0 4.8 9.3 2.4 3.2 nd 30.9 6.4 B nd
10.4 7.7 5.7 7.9 4.7 8.3 7.4 2.5 6.3 0.6 6.3 7.1 4.9 3.9 9.6 4.0 2.7 nd 30.9 6.5 B nd
PLB I PLB II
V. xanthoptera
9.9 6.7 5.6 7.3 5.0 7.6 6.4 3.7 6.8 2.7 6.3 7.3 5.0 3.7 9.3 3.5 3.0 0.3 31.8 nd A1 +++ Edema lethaltoxicity cardiotoxicity
LTc
V. basalis
b
Bold number shows a marked dierence from other species, nd: no detectable. na: no analysis. MDT: mandaratoxin. c LT: lethal toxin. d HO: hornetin. e Ant5: antigen 5. f Detected by SDS-PAGE.
a
Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Try Molecular weight (kDa)f Isoelectric point (pH) Type of enzyme Hemolysis Other properties identi®ed
Amino acids
Table 3 Amino acid compositions and physico-chemical properties of PLs from hornets and waspa
11.3 5.5 3.0 8.3 5.3 8.6 7.0 0 7.6 2.6 6.5 7.9 4.2 4.0 10.9 4.3 2.9 0 32.0 10.2 na +++ Edema neurotoxicity
HOd
V. ¯avitarsus
11.7 5.2 5.6 11.8 3.9 9.4 6.8 4.3 5.9 1.6 5.4 5.6 4.9 2.1 9.9 3.2 2.6 nd 27.0 nd nd nd Allergen
Ant5e
P. annularis
11.1 5.4 6.7 6.7 3.2 8.0 8.3 4.1 7.3 0.6 5.4 7.6 4.8 4.8 9.6 3.2 3.2 nd 37.0 nd B nd
PLB
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A2. Both puri®ed enzymes should be designated with a name such as Kumantoxin a and b to be associated with the traditional Japanese hornet name in the same way as many multifunctional enzymes simultaneously have particular toxin names. The puri®ed PLB a and b showed a catalytic or anity inclination on b and g residues of phosphatidylcholine. The PLB a had the higher anity for g position, but the PLB b had less anity for g position in our experiments. While, the amino acid compositions of PLBs from V. xanthoptera are compared to those from V. mandarinia, PLB a and b are corresponded to PLB I and PLB II, respectively (Table 3), despite a marked dierence between both kinetic properties, PLB I and PLB II were likely PLA1 type, but those of PLB a and b were strictly the types of PLB. We need more precise experiments to show whether the dierences of fatty acids residues in the b and g position in¯uence the enzymatic activity or not. The Indian plant, Aristolochia indica, is used for treating poisonous snake bites (Tsai et al., 1975). It has been found that aristolochic acid contained in the plant inhibits the activity of PLA2 from Viperid and Crotalid, and of other PLA2 from poisonous snakes (Vishwanath et al., 1987a; Vishwanath and Gowda, 1987b). The other alkaloids and plant isolates such as wedelolactone (from Eclipta prostrata ) (Melo et al., 1995), stigma sterol (from E.prostreta ), b-sitosterol (from E. prostrata ) and cabenegrin A-I and A-II (Cabeca-de negro) (Nakagawa et al., 1982) aect the inhibitory of PLA2. The Ki values of those anti PLA2 compounds were a similar range of cepharanthine from Stephania cepharantha (Table 2). Cepharanthine markedly inhibited the activities of PLB a and b from V. mandarinia, PLA2s from bee venom, Naja mossambica mossambica and bovine pancreas. The dierence of inhibitory actions between competitive or uncompetitive for hornet PLBs and noncompetitive for other PLA2s would suggest the characteristic dierences between PLB and PLA2. While the structurally similar compounds of berbamine, cycleanine, tetrandrine and isotetrandrine showed very low inhibitory activity. These results suggest that an analysis of the structural and catalitic correlation would provide useful knowledge for the enzymatic control of PLB. Acknowledgements We would like to thank Mr. Masao Chijimatsu of our institute for amino acid analysis. References Abe, T., Kawai, N., Niwa, A., 1982. Puri®cation and properties of a presynaptically active neurotoxin, mandaratoxin, from hornet (Vespa mandarinia ). Biochemistry 21, 1693±1697. Abe, T., Inamura, S., Akasu, M., 1991a. Eect of cepharanthin on the lethality and cardiovascular disorder by Mamushi, Agkistrodon halys blomhoi, snake venom. Folia Pharmacol (Japan) 98, 327± 336.
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