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Comparative study of amino acid composition in an extract from hornet venom sacs: High content of neuroactive amino acids in Vespa
Toslcwe Vol . 27, No . 6, pp. 683-688, 1989. Printed in Great Britain.
0041-0101/89 53.00+ .00 ~ 1989 Per~tnoo Pres plc
COMPARATIVE STUDY OF AMINO ACID COMPOSITION IN AN EXTRACT FROM HORNET VENOM SACS : HIGH CONTENT OF NEUROACTIVE AMINO ACIDS IN YESPA TAKASHI ABE, l YASUAKI HARIYA,l NOBUFUMI KAwAI2 and AxncO MIwA2
'Laboratory of Insect Toxicology, Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351, Japan; 'Department of Neurobiology, Tokyo Metropolitan Institute of Newosciences, Musashidai 2-6, Fuchu-shi, Tokyo 183, Japan (Accepted for publication 27 December 1988) T. ABE, Y. HARIYA, N. KAWAI and A. MIwA . Comparative study of amino acid composition in an extract from hornet venom sacs : high content of neuroactive amino acids in Vespa. Toxicon 27, 683-688, 1989 .-The amino acid compositions of extracts from the venom sacs of Vespa mandarmia, V. xanthoptera, V. tropica, V. analis and Vespula lewisi were analyzed . mAmino acids, which are inhibitory neurotransmitters, such as y-amino-nbutyric acid, taurine, ß-alanine and glycine, were predominant components in Vespa, but a minor component in Vespula. Glutamic acid, an aminergic excitatory neurotransmitter, was present in large quantities . Leucine, an insect autotoxin, was detected . Arginine was found in all the venoms and glutamine was also abundant . Tryptophan and histidine, precursors of serotonin and histamine, respectively, were also found . Thus, neuroactive amino acids that may exert inhibitory et%cts on insects neuronal axons and neuromuscular junctions were present in large amounts, and may facilitate paralysis of insect prey .
INTRODUCTION MANY xlxDS of insects serve as sources of food for hornets, which sting and paralyze them (ABE, 1985). The insects are then crushed by the hornet mandible and consumed. Since the minced meat is eaten, the venom in it must consist of orally nontoxic substances, perhaps substances such as biological amines, amino acids, peptides or proteins . To date, venom has been shown to contain amino acid derivatives such as histamine (Mikami and Abe, unpublished observations), serotonin (Mikami and Abe, unpublished observations) vespakinines (YASUHARA et al ., 1977), mastoparan (HIRAI et al . 1978) mandaratoxin (ABE et al., 1982), phospholipase A2 (ROSENBERG et al., 1977) and hornetin (Ho and Ko, 1986). The organ which produces venom is derived evolutionally from the reproductive organs . This suggests that venom, which was originally a secretion of the reproductive gland, should be rich in amino acids. In addition to an open circulatory system, insects possess a neuromuscular transmission mechanism dil%rent from that in mammals. Glutamic acid takes the place of acetylcholine at mammalian neuromuscular junctions (USHERWOOD et al., 1968 ; ABE et al., 1982). 683
68 4
T . ABE et al .
Leucine is an autotoxin that produces repetitive axonal excitation of insect nerve (TASHIRO et al., 1972). Analysis of the amino acid compositions of hornet venoms should provide some clarification of the fundamental role of amino acids in the venom. MATERIALS AND METHODS Materials Hornet species, Vespa mandarinéa, V. xanthoptera, V. tropics, V. analis and Vespula lewisi, were collected from the plains of Kanto in Japan . The venom sacs were obtained from the venom organs, frozen quickly and stored in liquid nitrogen until use . The numbers of hornets and average weight of each venom sac are listed in Table l . Sephadex G-50 was purchased from Pharnlacia Co. An amino acid calibration mixture containing Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, CyS, Met, Ile, Leu, Tyr, Phe, ammonium sulfate, Lys, His and Arg was a gift from Takara Kosan Co . The other chemicals used were as follows: phosphoserine (P-Ser), L-a-amino-n-butyric acid, 1methyl-l-hiaddine (1-McHis) and 3-methyl-l-hiatidine (3-McHis) from Sigma Chemical Co.; faunae (Tau), ethanolamine (EtAm) and y-amino-n-butyric acid (GABA) from Nakarai Chemical Co . ; phosphcethanol amine (P-EtAm), L-asparagine monohydrate (Aan), a-amino isobutyric acid (a-AiHA), ornithine HCI (Orn), histamine, acetylcholine iodide, noradrenaline and lithium chloride from Wako Chemical Co.; L-dioxylphenylacetic acid and o-phthaldehyde from Tokyo Kasei Chemical Co . ; Gln and Trp from Nippon Rikagaku Yakuhin Co .; ßalanine (ß-Ala) and lithium hydroxide monohydrate from Kanto Chemical Co . ; mercaptcethanol from Aldrich Chemical Co . ; dimethylsulfoxide from Dotaido Chemical Co . ; and serotonin creatinate from Merck . Preparation of crude venom sac extracts All procedures were carried out at 0~°C. Homogenates containing venom and its sac (25% in 50 mM acetate buffer, pH 5 .2) were prepared using a Polyfron homogenizer. The homogenates were centrifuged at 110,000 x g for 60 min, and the supernatants were used as crude venom sac preparations. Gel filtration of the crude venom sac extracts Supernatants were chromatographed at 4°C on a Sephadex G-50 column (~2 x 97 cm) equilibrated with 50 mM acetate buffer, pH 5 .2 . Three milliliters of each fraction were collected with an LKB Ultro Rac II fraction collector, monitored at 280 nm with a Pharmacia UV2 monitor . Glu and GABA were determined by the fluorescamine method (UDENFRn:ND et al., 1972) . Acetylcholine was measured by the hydroxamate method (IIESTRIN, 1949) . Analysis of physiological amino acids Thirty-three amino acids were analyzed by a JEOL 6AH automatic amino acid analyzer using a single column (¢0 .8 x 70 cm) packed with Hitachi 2613 resin. The first buffer was 44.5 mM lithium citrate, 66.1 mM LiCI and 6% ethanol, adjusted to pH 2 .80 with HCI. The second and third buffers were 100 mM lithium citrate at pH 3 .15 and 3 .92, respectively, adjusted with HCI . The fourth buffer was 217 mM lithium citrate and 650 mM LiCI, adjusted to pH 4 .55 with HCI . The first buffer was used for 180 min, followed by the second for 330 min, the third for 480 min and the fourth for 780 min . The column temperature was raised from 37°C to 60°C at 250 min. The flow rate was fixed at 0.8 ml per min . Fifty microliters of each sample was diluted to 1 ml in sample diluter (0 .2 N Li-citrate buffer, pH 2.2). The separated amino acids were analyzed by fluorescence intensity using a fluorescence indicator containing 0.8 g of aphthaldehyde, 2 ml mercaptcethanol and 10 ml of dimethylsulfoxide in 1 liter of 0.6 M borate buffer, pH 9.2 . The data were analyzed with a SIC 5000 integrator .
TABLE l . NUMBER AND WEIGHT OF HORNET VENOM SACS Hornet species Vespa mandarinia V. xanthoptera V. tropics V. analis Vespula lewisi
Number of venom sacs
Average weight of one venom sac (mg)
20 100 77 30 260
18 .0 3 .7 9 .l 4 .7 1 .58
Amino Acids in Hornet Venom Sacs
685
RESULTS
Sephadex G-SO column chromatography of the crude venom sac extract
Amino acids in the crude venom sac extract of each hornet were separated from high mol.wt compounds and a large amount of the serotonin by chromatography on a Sephadex G-50 column, as described in the Materials and Methods (see Fig. 1). High mol.wt compounds were commonly eluted in peak A (nos 20-25), corresponding to the void volume, and peak B (nos 2035), with mol.wts of a few tens of thousands. Peptides in the mol. wt range of a few thousand which absorb at 280 nm (peak C; nos 40-4 and
FIa. I . COLUMN CHROMATOGRAPHY OF CRUDE HORNET VENOMS ON SEPHADEX G-$l1. Absorbance at 280nm is shown by the solid (20 mm path length) and dotted lines (1 mm path length). Peak D (approx. nos 50-70) of each hornet venom was lyophilized for amino acid analysis. GABA, acetylcholine (ACh), histamine (Hst), Glu, noradrenaline (Nra) and serotonin (Srt) were chromatographed as standards, and determined by the methods of 8uorescamine (GABA, Hst and Glu), hydroxamate (ACh) or absorbance at 280 nm (Nra) as described in the Materials and Methods.
686
T . ABE et al.
whose existence was proposed in a previous study (Kawai et al., 1980 ; ABE and KAWAI, 1983) were found only in the case of V. analis . Lower mol. wt compounds, such as amino acids and biological amines were noted at peak D (nos 48-70) (KAWAI et al. 1980; AsE and KAWAI, 1983). Serotonin has an affinity for Sephadex and eluted from the column last, as peak E (nos 65-90) . Standard compounds were fractionated on the same column (Fig . l). GABA and acetylcholine eluted in fraction 52, Glu and histamine in fraction 56, and noradrenaline in fraction 60. Peak D fractions were lyophilized and then dissolved in distilled water to a volume of 2 ml for amino acid analysis. Amino acid composition of hornet venom sac extract In the venom extract, P-Ser, Tau, Asp, Thr, Ser, Asn, Glu, Gln, Gly, Ala, Val, Met, DOPA, Ile, Leu, Tyr, Phe, ß-Ala, GABA, EtAm, Orn, Trp, His, 1-McHis, 3-MeHis and Arg were detected (Table 2). Each venom sac from V. mandarinia contained approximately 963 nmoles of amino acids; V, tropica, V. analis, V. xanthoptera and Vespula lewisi contained approximately 546, 372, 206 and 72 nmoles of amino acids, respectively . Glu was usually the most abundant amino acid at over 50 mole % . In the case of V. mandarinia, Arg, Gln, Tau, GABA, Gly and His were present at more than 2 mole %.For V. analis, the percent composition was virtually the same as that of V. mandarinia, with Arg, Tau, Gln, Gly and GABA being present in greater amounts. Based on this essentially similar composition, V. analis may be considered a morphological miniature of V. manderinia ; that is, both are taxonomic near relatives. In both V. tropica and V. xanthoptera, Arg, Gln, Tau, Leu, Gly, Ala and GABA were rather abundant, as was also the case for the other hornets. As in the Vespa species, Vespula lewisi contained large amounts of Glu, Ala, Leu, Gln, Trp, Arg and Gly; however, ß-Ala, GABA and Tau were present in very small amounts while Ser, Val and Ile were more abundant than in Vespa. The presence of those neuroactive amino acids is advantageous for suppressing the nerves of stung insects. DISCUSSION
In the present study, identification has been made of amino acids obtained from the venom sacs of various hornet species. Our results differ fundamentally from those in a preliminary short report (IxAN and IsxnY, 1973). Among the amino acids present in abundance in Yespa and Vespula, the Glu content is extremely high . Glu is recognized as a neurotransmitter at aminergic neuromuscular junctions such as insect and arthropod synapses (Ust-tEttwoon et al., 1968 ; AsE et al., 1983). GABA, ß-Ala, Tau and Gly, the socalled co-amino acids, are inhibitory neurotransmitters (CtntTts and WAr.xnvs, 1965). Thus, excitatory and inhibitory transmitters are predominantly present in hornet venom. Excess amounts of these transmitters cause paralysis in houseflies (unpublished data from our laboratory). It appears quite likely that amino acids promote paralysis of insects into which venom has been injected. In our previous study, serotonin and histamine were found as major components in hornet venom (Mikami and Abe, unpublished observations) . This study (Fig. 1) and also our previous reports (KAWAt et al., 1980; AsE and Knwnt, 1983) show a high content of serotonin in several hornet venoms as determined by gel filtration on Sephadex G-50. Serotonin and histamine are produced by the enzymatic decarboxylation of Trp and His, respectively, thus both amino acids probably leak from the venom gland. Leu is an autotoxin secreted from insect nerves during repetitive axonal response (Tnsxtxo et al .,
3 .07 48 .60 4 .67 9 .00 12 .48 6.53 479 .52 49 .80 30.96 54.96 15 .12 ' ' 8 .97 24.48 16 .92 15 .00 12 .00 42 .48 13 .68 ' 18 .48 20 .28 0 .45 0 .09 75 .57
963 .11
P-Ser Tau Asp Thr Ser Asn Glu Gln Gly Ala Val Met DOPA Ile Lue Tyr Phe ß-Als GABA EtAm Orn Trp His 1-MeHis 3-MeHis Arg
Total
100 .02
0.32 5 .05 0.48 0.93 1 .30 0 .68 49 .79 5 .17 3 .21 5 .71 1 .57 0 .93 2 .54 1 .76 1 .56 1 .25 4 .41 1 .42 1 .92 2 .11 0 .05 0.01 7 .85
mole%
'Less than 0 .05 nmoles/venom sac .
nmoles/ venom sac
Amino acids
V. mandarinia
2.
206.31
0 .98 7 .94 3 .18 3 .11 4 .71 1 .71 100 .89 10 .56 6 .07 1 .73 4.71 5 .18 " 3.16 6.83 1 .34 2.30 5.25 2 .59 1 .87 0.41 5 .63 2.21 0.05 ' 23 .90
nmoles/ venom sac
99.99
0 .47 3 .85 1 .54 1 .51 .2 .28 0 .83 48 .90 5 .12 2 .94 0 .84 2.28 2 .51 1 .53 3 .31 0 .65 1 .11 2.55 1 .26 0.91 0.20 2.73 1 .07 0.02 11 .58
mole%
545 .84
2.83 19.35 3.37 4.69 6 .79 9.47 292.90 15 .11 12.56 29 .30 7.22 1 .75 0.54 29.43 28 .15 6.91 21 .96 3 .96 1 .70 2.69 ' 12 .68 2.85 1 .18 ' 28 .09
nmoles/ venom sac
V. tropics
99.94
0.52 3.55 0.68 0.86 1 .24 1 .73 53.66 2.77 2.30 5.37 1 .32 0.32 0.10 5.39 5.16 1 .27 4.02 0.73 0.31 0.49 2.32 0.52 0.16 5.15
mole%
V. analir
0 .32 6 .21 0 .41 0 .75 1 .19 69 .82 2 .95 1 .28 3 .95 0 .59 0 .62 1 .16 0 .49 0 .64 0 .30 3 .44 0 .71 1 .09 4 .05 99 .97
372 .48
mole%
1 .19 23 .13 1 .54 2 .81 4 .42 ' 260 .07 11 .00 4 .76 14 .73 2 .21 ' ' 2 .31 4 .33 1 .83 2 .40 1 .13 12.81 2 .64 ' ' 4 .07 " ' 15 .10
nmoles/ venom sac
AMINO ACID COMPOSITION OF HORNET YENOM EXTRACTS
V. xanthoptera
TABLE
72 .22
0 .48 0 .57 1 .37 0 .99 3 .19 1 .19 33 .71 3 .76 1 .77 6 .77 1 .89 0 .09 ' 1 .97 4.68 0 .47 1 .07 0.25 0 .47 0 .91 0 .93 3 .31 0 .49 0 .15 0 .15 1 .58
nmoles/ venom sac
99 .96
0 .66 0 .79 1 .89 1 .37 4 .42 1 .65 46 .67 5 .21 2 .45 9 .38 2 .61 0 .13 2 .72 6 .48 0 .65 1 .48 0 .35 0 .66 1 .26 1 .29 4 .59 0 .68 0 .20 0 .21 2 .19
mole%
Veapaia lewisi
c3
c ô:°°
y
688
T. ABE et al .
1972), that paralyzes neuronal transmission . Leu in venom will thus exert an inhibitory effect on the nerves of insects. The patent capillary circulatory system of insects enhances the neural effects of amino acids, because insect neural organs floating in hemolymph are affected more quickly and directly . In mammals, however, venom amino acids are rapidly metabolized in the liver. A venom extract may actually be a mixture of venom and the sac itself. Therefore, the question of how many amino acids are extracted from the sac is pertinent . The amino acid composition of insect plasma is well known. In many insect, those concentrations (except glutamine) are a few nmoles per mg plasma (WoonxnvG, 1985) . Insect tissues and plasma show probably a similar range of amino acids. In this study, neuroactive acids found in hornet venom sac extracts are present in higher concentration than other amino acids which are estimated at a few nmoles per mg of whole venom sac. This suggests that only small amounts of amino acids come from the sac itself. Acknowledgements-We thank Mr Snneo Futuna and Mr SATOSHt TAtcrrA for their assistance in hornet collection .
REFERENCES Ast~ T. (1985) Hornet venoms : a general nature of the hornet, a property of venom and a stinging damage. Honeybee Sci. 6, 13 . Ast:, T. and Kwwet, N. (1983) Catdioactive effects of hornet venom (Vespa martdarinia) . Comp . Biochem. Physiol. 76C, 221 . AsE, T., Kwwnt, N. and NtW~, A. (1982) Purification and properties of a presynaptically acting neurotoxin, mandaratoxin, from hornet (Vespa mandarinia). Biochemistry 21, 1693 . Ast:, T., Kwwnt, N. and Mtwe, A. (1983) Effects of a spider toxin on the glutaminergic synapse of lobster muscle . !. PhysioJ. 339, 243. Cusps, C. R. and W~u.xtxs, J. C. (1965) The pharmacology of amino acids related to gamma-amino butyric acid. Pharmac. Rev. 17, 347. HFSranv, S. (1949) The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J. biol. Chem . 180, 249. Httw, Y., Yesuttew~, T., Yostnne, H. and N~twnw+, T. (1978) A new mast cell degrading peptide "mastoparan" in wasp venom. Pept. Chem . 155. Ho, C. L. and Ko, J. L. (1986) Hornetin : the lethal protein of the hornet (Vespa Jlavitarsus) venom. FEBS Lett. 209, 18 . Itcwx, R. and Isttw, J. (1973) Free amino acids in the haemolymph and the venom of the oriental hornet (Vespa orientalis) . Comp . Biochem. Physiol. 44B, 949. Knw~t, N., Ast?, T., How, S. and Ntwn, A. (1980) Effects of neurotoxin in hornet venom on neuromuscular junction of lobster . Comp . Biochem. Physiol. 65C, 87 . RosertssxG, P., Isttev, Y. and $1MON, G. (1977) Phospholipase A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxieon 15, 141 . T~stnx0, S., T~nauct-n, E. and E'ro, M. (1972) Isolation of a neuroactive substance, t .-leucine, from the blood of silkworm poisoned with DDT. Agric. Biol. Chem. 36, 2465 . UnexFxtErto, S., $TIIN, S., Bottt,etv, P., DAIItMAN, W., Ltastoxuses, M. and Wvroet .i:, M . (1972) Application of fluorescamine: a new reagent for assay of amino acids, peptides, proteins and other primary amines in the picomole range. Science, N. Y. 178, 871 . Ust~rewooo, P. N. R., Mtctnu, P. and L&~F, G. (1968) L-Glutamate at insect excitatory nerve-muscle synapses, Nature 219, 1169 . YASUFiARA, T., Yosttwe, H. and NAKAIIMA, T. (1977) Chemical investigation of the hornet (Vespa xanthoptera Cameron) venom. The structure of a new bradykinin analog "Vespakinin-X" . Chem . Pharmac. Bull. 25, 936, Woooruxc, J . P. (1985) Circulatory systems. In : Fundamentals of Invect Physiology, pp . 46 9 (GLUM, M. S., Ed .). London : John Wiley.
0041-0101/89 53.00+ .00 ~ 1989 Per~tnoo Pres plc
COMPARATIVE STUDY OF AMINO ACID COMPOSITION IN AN EXTRACT FROM HORNET VENOM SACS : HIGH CONTENT OF NEUROACTIVE AMINO ACIDS IN YESPA TAKASHI ABE, l YASUAKI HARIYA,l NOBUFUMI KAwAI2 and AxncO MIwA2
'Laboratory of Insect Toxicology, Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama 351, Japan; 'Department of Neurobiology, Tokyo Metropolitan Institute of Newosciences, Musashidai 2-6, Fuchu-shi, Tokyo 183, Japan (Accepted for publication 27 December 1988) T. ABE, Y. HARIYA, N. KAWAI and A. MIwA . Comparative study of amino acid composition in an extract from hornet venom sacs : high content of neuroactive amino acids in Vespa. Toxicon 27, 683-688, 1989 .-The amino acid compositions of extracts from the venom sacs of Vespa mandarmia, V. xanthoptera, V. tropica, V. analis and Vespula lewisi were analyzed . mAmino acids, which are inhibitory neurotransmitters, such as y-amino-nbutyric acid, taurine, ß-alanine and glycine, were predominant components in Vespa, but a minor component in Vespula. Glutamic acid, an aminergic excitatory neurotransmitter, was present in large quantities . Leucine, an insect autotoxin, was detected . Arginine was found in all the venoms and glutamine was also abundant . Tryptophan and histidine, precursors of serotonin and histamine, respectively, were also found . Thus, neuroactive amino acids that may exert inhibitory et%cts on insects neuronal axons and neuromuscular junctions were present in large amounts, and may facilitate paralysis of insect prey .
INTRODUCTION MANY xlxDS of insects serve as sources of food for hornets, which sting and paralyze them (ABE, 1985). The insects are then crushed by the hornet mandible and consumed. Since the minced meat is eaten, the venom in it must consist of orally nontoxic substances, perhaps substances such as biological amines, amino acids, peptides or proteins . To date, venom has been shown to contain amino acid derivatives such as histamine (Mikami and Abe, unpublished observations), serotonin (Mikami and Abe, unpublished observations) vespakinines (YASUHARA et al ., 1977), mastoparan (HIRAI et al . 1978) mandaratoxin (ABE et al., 1982), phospholipase A2 (ROSENBERG et al., 1977) and hornetin (Ho and Ko, 1986). The organ which produces venom is derived evolutionally from the reproductive organs . This suggests that venom, which was originally a secretion of the reproductive gland, should be rich in amino acids. In addition to an open circulatory system, insects possess a neuromuscular transmission mechanism dil%rent from that in mammals. Glutamic acid takes the place of acetylcholine at mammalian neuromuscular junctions (USHERWOOD et al., 1968 ; ABE et al., 1982). 683
68 4
T . ABE et al .
Leucine is an autotoxin that produces repetitive axonal excitation of insect nerve (TASHIRO et al., 1972). Analysis of the amino acid compositions of hornet venoms should provide some clarification of the fundamental role of amino acids in the venom. MATERIALS AND METHODS Materials Hornet species, Vespa mandarinéa, V. xanthoptera, V. tropics, V. analis and Vespula lewisi, were collected from the plains of Kanto in Japan . The venom sacs were obtained from the venom organs, frozen quickly and stored in liquid nitrogen until use . The numbers of hornets and average weight of each venom sac are listed in Table l . Sephadex G-50 was purchased from Pharnlacia Co. An amino acid calibration mixture containing Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, CyS, Met, Ile, Leu, Tyr, Phe, ammonium sulfate, Lys, His and Arg was a gift from Takara Kosan Co . The other chemicals used were as follows: phosphoserine (P-Ser), L-a-amino-n-butyric acid, 1methyl-l-hiaddine (1-McHis) and 3-methyl-l-hiatidine (3-McHis) from Sigma Chemical Co.; faunae (Tau), ethanolamine (EtAm) and y-amino-n-butyric acid (GABA) from Nakarai Chemical Co . ; phosphcethanol amine (P-EtAm), L-asparagine monohydrate (Aan), a-amino isobutyric acid (a-AiHA), ornithine HCI (Orn), histamine, acetylcholine iodide, noradrenaline and lithium chloride from Wako Chemical Co.; L-dioxylphenylacetic acid and o-phthaldehyde from Tokyo Kasei Chemical Co . ; Gln and Trp from Nippon Rikagaku Yakuhin Co .; ßalanine (ß-Ala) and lithium hydroxide monohydrate from Kanto Chemical Co . ; mercaptcethanol from Aldrich Chemical Co . ; dimethylsulfoxide from Dotaido Chemical Co . ; and serotonin creatinate from Merck . Preparation of crude venom sac extracts All procedures were carried out at 0~°C. Homogenates containing venom and its sac (25% in 50 mM acetate buffer, pH 5 .2) were prepared using a Polyfron homogenizer. The homogenates were centrifuged at 110,000 x g for 60 min, and the supernatants were used as crude venom sac preparations. Gel filtration of the crude venom sac extracts Supernatants were chromatographed at 4°C on a Sephadex G-50 column (~2 x 97 cm) equilibrated with 50 mM acetate buffer, pH 5 .2 . Three milliliters of each fraction were collected with an LKB Ultro Rac II fraction collector, monitored at 280 nm with a Pharmacia UV2 monitor . Glu and GABA were determined by the fluorescamine method (UDENFRn:ND et al., 1972) . Acetylcholine was measured by the hydroxamate method (IIESTRIN, 1949) . Analysis of physiological amino acids Thirty-three amino acids were analyzed by a JEOL 6AH automatic amino acid analyzer using a single column (¢0 .8 x 70 cm) packed with Hitachi 2613 resin. The first buffer was 44.5 mM lithium citrate, 66.1 mM LiCI and 6% ethanol, adjusted to pH 2 .80 with HCI. The second and third buffers were 100 mM lithium citrate at pH 3 .15 and 3 .92, respectively, adjusted with HCI . The fourth buffer was 217 mM lithium citrate and 650 mM LiCI, adjusted to pH 4 .55 with HCI . The first buffer was used for 180 min, followed by the second for 330 min, the third for 480 min and the fourth for 780 min . The column temperature was raised from 37°C to 60°C at 250 min. The flow rate was fixed at 0.8 ml per min . Fifty microliters of each sample was diluted to 1 ml in sample diluter (0 .2 N Li-citrate buffer, pH 2.2). The separated amino acids were analyzed by fluorescence intensity using a fluorescence indicator containing 0.8 g of aphthaldehyde, 2 ml mercaptcethanol and 10 ml of dimethylsulfoxide in 1 liter of 0.6 M borate buffer, pH 9.2 . The data were analyzed with a SIC 5000 integrator .
TABLE l . NUMBER AND WEIGHT OF HORNET VENOM SACS Hornet species Vespa mandarinia V. xanthoptera V. tropics V. analis Vespula lewisi
Number of venom sacs
Average weight of one venom sac (mg)
20 100 77 30 260
18 .0 3 .7 9 .l 4 .7 1 .58
Amino Acids in Hornet Venom Sacs
685
RESULTS
Sephadex G-SO column chromatography of the crude venom sac extract
Amino acids in the crude venom sac extract of each hornet were separated from high mol.wt compounds and a large amount of the serotonin by chromatography on a Sephadex G-50 column, as described in the Materials and Methods (see Fig. 1). High mol.wt compounds were commonly eluted in peak A (nos 20-25), corresponding to the void volume, and peak B (nos 2035), with mol.wts of a few tens of thousands. Peptides in the mol. wt range of a few thousand which absorb at 280 nm (peak C; nos 40-4 and
FIa. I . COLUMN CHROMATOGRAPHY OF CRUDE HORNET VENOMS ON SEPHADEX G-$l1. Absorbance at 280nm is shown by the solid (20 mm path length) and dotted lines (1 mm path length). Peak D (approx. nos 50-70) of each hornet venom was lyophilized for amino acid analysis. GABA, acetylcholine (ACh), histamine (Hst), Glu, noradrenaline (Nra) and serotonin (Srt) were chromatographed as standards, and determined by the methods of 8uorescamine (GABA, Hst and Glu), hydroxamate (ACh) or absorbance at 280 nm (Nra) as described in the Materials and Methods.
686
T . ABE et al.
whose existence was proposed in a previous study (Kawai et al., 1980 ; ABE and KAWAI, 1983) were found only in the case of V. analis . Lower mol. wt compounds, such as amino acids and biological amines were noted at peak D (nos 48-70) (KAWAI et al. 1980; AsE and KAWAI, 1983). Serotonin has an affinity for Sephadex and eluted from the column last, as peak E (nos 65-90) . Standard compounds were fractionated on the same column (Fig . l). GABA and acetylcholine eluted in fraction 52, Glu and histamine in fraction 56, and noradrenaline in fraction 60. Peak D fractions were lyophilized and then dissolved in distilled water to a volume of 2 ml for amino acid analysis. Amino acid composition of hornet venom sac extract In the venom extract, P-Ser, Tau, Asp, Thr, Ser, Asn, Glu, Gln, Gly, Ala, Val, Met, DOPA, Ile, Leu, Tyr, Phe, ß-Ala, GABA, EtAm, Orn, Trp, His, 1-McHis, 3-MeHis and Arg were detected (Table 2). Each venom sac from V. mandarinia contained approximately 963 nmoles of amino acids; V, tropica, V. analis, V. xanthoptera and Vespula lewisi contained approximately 546, 372, 206 and 72 nmoles of amino acids, respectively . Glu was usually the most abundant amino acid at over 50 mole % . In the case of V. mandarinia, Arg, Gln, Tau, GABA, Gly and His were present at more than 2 mole %.For V. analis, the percent composition was virtually the same as that of V. mandarinia, with Arg, Tau, Gln, Gly and GABA being present in greater amounts. Based on this essentially similar composition, V. analis may be considered a morphological miniature of V. manderinia ; that is, both are taxonomic near relatives. In both V. tropica and V. xanthoptera, Arg, Gln, Tau, Leu, Gly, Ala and GABA were rather abundant, as was also the case for the other hornets. As in the Vespa species, Vespula lewisi contained large amounts of Glu, Ala, Leu, Gln, Trp, Arg and Gly; however, ß-Ala, GABA and Tau were present in very small amounts while Ser, Val and Ile were more abundant than in Vespa. The presence of those neuroactive amino acids is advantageous for suppressing the nerves of stung insects. DISCUSSION
In the present study, identification has been made of amino acids obtained from the venom sacs of various hornet species. Our results differ fundamentally from those in a preliminary short report (IxAN and IsxnY, 1973). Among the amino acids present in abundance in Yespa and Vespula, the Glu content is extremely high . Glu is recognized as a neurotransmitter at aminergic neuromuscular junctions such as insect and arthropod synapses (Ust-tEttwoon et al., 1968 ; AsE et al., 1983). GABA, ß-Ala, Tau and Gly, the socalled co-amino acids, are inhibitory neurotransmitters (CtntTts and WAr.xnvs, 1965). Thus, excitatory and inhibitory transmitters are predominantly present in hornet venom. Excess amounts of these transmitters cause paralysis in houseflies (unpublished data from our laboratory). It appears quite likely that amino acids promote paralysis of insects into which venom has been injected. In our previous study, serotonin and histamine were found as major components in hornet venom (Mikami and Abe, unpublished observations) . This study (Fig. 1) and also our previous reports (KAWAt et al., 1980; AsE and Knwnt, 1983) show a high content of serotonin in several hornet venoms as determined by gel filtration on Sephadex G-50. Serotonin and histamine are produced by the enzymatic decarboxylation of Trp and His, respectively, thus both amino acids probably leak from the venom gland. Leu is an autotoxin secreted from insect nerves during repetitive axonal response (Tnsxtxo et al .,
3 .07 48 .60 4 .67 9 .00 12 .48 6.53 479 .52 49 .80 30.96 54.96 15 .12 ' ' 8 .97 24.48 16 .92 15 .00 12 .00 42 .48 13 .68 ' 18 .48 20 .28 0 .45 0 .09 75 .57
963 .11
P-Ser Tau Asp Thr Ser Asn Glu Gln Gly Ala Val Met DOPA Ile Lue Tyr Phe ß-Als GABA EtAm Orn Trp His 1-MeHis 3-MeHis Arg
Total
100 .02
0.32 5 .05 0.48 0.93 1 .30 0 .68 49 .79 5 .17 3 .21 5 .71 1 .57 0 .93 2 .54 1 .76 1 .56 1 .25 4 .41 1 .42 1 .92 2 .11 0 .05 0.01 7 .85
mole%
'Less than 0 .05 nmoles/venom sac .
nmoles/ venom sac
Amino acids
V. mandarinia
2.
206.31
0 .98 7 .94 3 .18 3 .11 4 .71 1 .71 100 .89 10 .56 6 .07 1 .73 4.71 5 .18 " 3.16 6.83 1 .34 2.30 5.25 2 .59 1 .87 0.41 5 .63 2.21 0.05 ' 23 .90
nmoles/ venom sac
99.99
0 .47 3 .85 1 .54 1 .51 .2 .28 0 .83 48 .90 5 .12 2 .94 0 .84 2.28 2 .51 1 .53 3 .31 0 .65 1 .11 2.55 1 .26 0.91 0.20 2.73 1 .07 0.02 11 .58
mole%
545 .84
2.83 19.35 3.37 4.69 6 .79 9.47 292.90 15 .11 12.56 29 .30 7.22 1 .75 0.54 29.43 28 .15 6.91 21 .96 3 .96 1 .70 2.69 ' 12 .68 2.85 1 .18 ' 28 .09
nmoles/ venom sac
V. tropics
99.94
0.52 3.55 0.68 0.86 1 .24 1 .73 53.66 2.77 2.30 5.37 1 .32 0.32 0.10 5.39 5.16 1 .27 4.02 0.73 0.31 0.49 2.32 0.52 0.16 5.15
mole%
V. analir
0 .32 6 .21 0 .41 0 .75 1 .19 69 .82 2 .95 1 .28 3 .95 0 .59 0 .62 1 .16 0 .49 0 .64 0 .30 3 .44 0 .71 1 .09 4 .05 99 .97
372 .48
mole%
1 .19 23 .13 1 .54 2 .81 4 .42 ' 260 .07 11 .00 4 .76 14 .73 2 .21 ' ' 2 .31 4 .33 1 .83 2 .40 1 .13 12.81 2 .64 ' ' 4 .07 " ' 15 .10
nmoles/ venom sac
AMINO ACID COMPOSITION OF HORNET YENOM EXTRACTS
V. xanthoptera
TABLE
72 .22
0 .48 0 .57 1 .37 0 .99 3 .19 1 .19 33 .71 3 .76 1 .77 6 .77 1 .89 0 .09 ' 1 .97 4.68 0 .47 1 .07 0.25 0 .47 0 .91 0 .93 3 .31 0 .49 0 .15 0 .15 1 .58
nmoles/ venom sac
99 .96
0 .66 0 .79 1 .89 1 .37 4 .42 1 .65 46 .67 5 .21 2 .45 9 .38 2 .61 0 .13 2 .72 6 .48 0 .65 1 .48 0 .35 0 .66 1 .26 1 .29 4 .59 0 .68 0 .20 0 .21 2 .19
mole%
Veapaia lewisi
c3
c ô:°°
y
688
T. ABE et al .
1972), that paralyzes neuronal transmission . Leu in venom will thus exert an inhibitory effect on the nerves of insects. The patent capillary circulatory system of insects enhances the neural effects of amino acids, because insect neural organs floating in hemolymph are affected more quickly and directly . In mammals, however, venom amino acids are rapidly metabolized in the liver. A venom extract may actually be a mixture of venom and the sac itself. Therefore, the question of how many amino acids are extracted from the sac is pertinent . The amino acid composition of insect plasma is well known. In many insect, those concentrations (except glutamine) are a few nmoles per mg plasma (WoonxnvG, 1985) . Insect tissues and plasma show probably a similar range of amino acids. In this study, neuroactive acids found in hornet venom sac extracts are present in higher concentration than other amino acids which are estimated at a few nmoles per mg of whole venom sac. This suggests that only small amounts of amino acids come from the sac itself. Acknowledgements-We thank Mr Snneo Futuna and Mr SATOSHt TAtcrrA for their assistance in hornet collection .
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