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Makatoxin I, a Novel Toxin Isolated from the Venom of the Scorpion Buthus martensi Karsch, Exhibits Nitrergic Actions

1997; Elsevier BV; Volume: 272; Issue: 13 Linguagem: Inglês

10.1074/jbc.272.13.8320

1083-351X

Jianping Gong, R. Manjunatha Kini, M.C.E. Gwee, P. Gopalakrishnakone, M.C.M. Chung,

Nicotinic Acetylcholine Receptors Study

Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 ± 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 °C, 5% CO2 in O2). MkTx I (2 μg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nω-nitro-L-arginine methyl ester (50 μM). Thus, MkTx I is the first α-toxin that can mediate nitrergic responses in the rat isolated ACM. Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 ± 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 °C, 5% CO2 in O2). MkTx I (2 μg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nω-nitro-L-arginine methyl ester (50 μM). Thus, MkTx I is the first α-toxin that can mediate nitrergic responses in the rat isolated ACM. INTRODUCTIONScorpion venoms consist of complex mixtures of several toxins that exhibit various pharmacological activities including selective actions at sodium and potassium channels, which are the major molecular targets of scorpion toxins (1Mebs D. Hucho F. Shier W.T. Mebs D. Handbook of Toxinology. Marcel Dekker, New York1990: 493-600Google Scholar). The scorpion Buthus martensi Karsch is widely distributed in China (2Song D.X. Lu X.X. Shang J.W. Bulletin of Biology (China). 1982; 1: 22-25Google Scholar), but relatively few reports have been published on the biological properties of its venom (MKV) 1The abbreviations used are:MKVButhus martensi Karsch venomMkTx Imakatoxin IACManococcygeus muscleL-NAMENω-nitro-L-arginine methyl esterNOnitric oxideFSfield stimulation. or toxins. Electrophysiological studies indicate that MKV contains some neurotoxic compounds that possibly act on the Na1+ channels in the excitable membrane of nerve and muscle (3Shih Y.L. Xu Y.Z. Xu K. Acta Physiol. Sin. 1982; 34: 428-433Google Scholar), cultured mouse cardiomyocyte (4Qi H. Zhong G.G. Chen L. Jiang Y. Wei J.J. Acta Pharmacol. Sin. 1993; 14: 361-364Google Scholar), and rat anterior pituitary cells (5Bauer C.K. Krylov B. Zhou P.A. Schwarz J.R. Toxicon. 1992; 30: 581-589Crossref PubMed Scopus (3) Google Scholar).In a preliminary study we observed that MKV produced relaxant responses of the rat precontracted anococcygeus muscle (ACM), suggesting that some constituent toxin(s) present in MKV could mimic the effects of nitrergic transmission involving the release of the inhibitory neurotransmitter nitric oxide (NO). 2J. P. Gong, M. C. E. Gwee, and P. Gopalakrishnakone, unpublished observation. Therefore, we were interested in isolating and purifying the bioactive component(s) present in the venom as well as identifying and characterizing the pure toxin(s) mediating the relaxant responses of the rat ACM. In this communication we report the isolation, purification, and complete amino acid sequence of a novel toxin, MkTx I, isolated from the venom of B. martensi Karsch and provide the first documentation of some novel nitrergic actions mediated by a scorpion toxin.DISCUSSIONMkTx I is structurally homologous to other scorpion toxins so far described: (a) it consists of one single chain of 64 residues cross-linked by 4 disulfide bridges; and (b) it lacks methionine, phenylalanine, and histidine. MkTx I belongs to the α-type scorpion toxin group; it has a short J-loop (from Cys-16 to Cys-22) and a long B-loop (From Cys-36 to Cys-46) (14Simard J.M. Watt D.D. Polis G.A. The Biology of Scorpions. Stanford University Press, Stanford, CA1990: 414-444Google Scholar). Furthermore, it displayed a very high similarity with toxins of group 3 (Fig. 4B) according to the classification of Rochat et al. (15Rochat H. Bernard P. Couraud F. Adv. Cytopharmacol. 1979; 3: 325-334PubMed Google Scholar). In this group, MkTx I has highest homology with Bot II from North African scorpion B. occitanus tunetanus venom. However, it differed significantly from those of β-type neurotoxins isolated from the venoms of North American and South American scorpions. For example, Css II showed only 25% similarity with MkTx I. Moreover, MkTx I has 5 aspartic acid and 5 asparagine residues with 1 glutamic acid and 1 glutamine residue, in contrast to β-type scorpion toxins that contain more glutamic acid and glutamine residues than aspartic acid and asparagine residues (16Mcintosh M.E. Watt D.D. De Vries A. Kochva E. Toxins of Animal and Plant Origin. Gordon and Breach, New York1973: 529-544Google Scholar, 17Possani D.L. Fletcher L.P. Alagon B.C.A. Alagon A.C. Julia Z.J. Toxicon. 1980; 18: 175-183Crossref PubMed Scopus (25) Google Scholar).The marked relaxant responses of the carbachol-precontracted ACM to MkTx I, like the nitrergic (relaxant) responses to FS, were effectively blocked by 50 μM L-NAME, a stereoselective inhibitor of the enzyme nitric oxide synthase involved in the synthesis of NO from L-arginine and oxygen (18Moncada S. Palmer R.M.J. Higgs E.A. Biochem. Pharmacol. 1989; 38: 1709-1715Crossref PubMed Scopus (1097) Google Scholar, 19Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). The relaxant responses of the ACM produced by MkTx I are therefore also likely to be NO-mediated because there is now strong evidence that implicates NO as the inhibitory neurotransmitter involved in nitrergic transmission (20Li C.G. Rand M.J. Clin. Exp. Pharmacol. Physiol. 1989; 16: 933-938Crossref PubMed Scopus (166) Google Scholar) and, consequently, in mediating the relaxant responses of the ACM (21Martin W. Gillespie J.S. Bell C. Novel Peripheral Neurotransmitters. Pergamon, New York1991: 65-79Google Scholar, 22Rand M.J. Clin. Exp. Pharmac. Physiol. 1992; 19: 147-169Crossref PubMed Scopus (362) Google Scholar). Unlike the relaxant responses of the ACM to FS and MkTx I, the relaxant responses of the ACM to sodium nitroprusside, an agent that directly releases its NO via metabolism (23Kowaluk E.A. Seth P. Fung H.L. J. Pharmacol. Exp. Ther. 1992; 262: 916-922PubMed Google Scholar), were not blocked by L-NAME. This provides further evidence for the involvement of endogenous NO released prejunctionally in mediating the relaxant responses of the ACM to MkTx I.In conclusion, a novel toxin MkTx I with nitrergic actions has been purified from the venom of the scorpion B. martensi Karsch, and the novel nitrergic actions of MKV in the rat isolated anococcygeus muscle can be attributed, at least in part, to the actions of the pure toxin MkTx I present in the venom. INTRODUCTIONScorpion venoms consist of complex mixtures of several toxins that exhibit various pharmacological activities including selective actions at sodium and potassium channels, which are the major molecular targets of scorpion toxins (1Mebs D. Hucho F. Shier W.T. Mebs D. Handbook of Toxinology. Marcel Dekker, New York1990: 493-600Google Scholar). The scorpion Buthus martensi Karsch is widely distributed in China (2Song D.X. Lu X.X. Shang J.W. Bulletin of Biology (China). 1982; 1: 22-25Google Scholar), but relatively few reports have been published on the biological properties of its venom (MKV) 1The abbreviations used are:MKVButhus martensi Karsch venomMkTx Imakatoxin IACManococcygeus muscleL-NAMENω-nitro-L-arginine methyl esterNOnitric oxideFSfield stimulation. or toxins. Electrophysiological studies indicate that MKV contains some neurotoxic compounds that possibly act on the Na1+ channels in the excitable membrane of nerve and muscle (3Shih Y.L. Xu Y.Z. Xu K. Acta Physiol. Sin. 1982; 34: 428-433Google Scholar), cultured mouse cardiomyocyte (4Qi H. Zhong G.G. Chen L. Jiang Y. Wei J.J. Acta Pharmacol. Sin. 1993; 14: 361-364Google Scholar), and rat anterior pituitary cells (5Bauer C.K. Krylov B. Zhou P.A. Schwarz J.R. Toxicon. 1992; 30: 581-589Crossref PubMed Scopus (3) Google Scholar).In a preliminary study we observed that MKV produced relaxant responses of the rat precontracted anococcygeus muscle (ACM), suggesting that some constituent toxin(s) present in MKV could mimic the effects of nitrergic transmission involving the release of the inhibitory neurotransmitter nitric oxide (NO). 2J. P. Gong, M. C. E. Gwee, and P. Gopalakrishnakone, unpublished observation. Therefore, we were interested in isolating and purifying the bioactive component(s) present in the venom as well as identifying and characterizing the pure toxin(s) mediating the relaxant responses of the rat ACM. In this communication we report the isolation, purification, and complete amino acid sequence of a novel toxin, MkTx I, isolated from the venom of B. martensi Karsch and provide the first documentation of some novel nitrergic actions mediated by a scorpion toxin.