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Functional multivalence and structural uniqueness of a new polypeptide from Chinese scorpion Buthus martensi Karsch
Abstracts
493
BmK AS, a bioactive polypeptide composed of 63 amino acids purified from the venom of Chinese scorpion Bufhus martensi Karsch (BmK) has been demonstrated to be a potent activator of the ryanodine receptor, and also enhances noradrenaline release and Na+ conductance. Here we report another BmK AS-like polypeptide from the same venom. The BmK venom was isolated by a series of courses, including gel filtration on Sephadex G-25, Sephadex G-50 columns and anion-exchange chromatography on a DEAE-Sephadex A-50 column. Finally, a new polypeptide was purified by repeated high-performance liquid chromatography (HPLC) and examined as a band in slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. The retention time of the purified polypeptide was quite different to that of the known BmK AS in the HPLC pattern. The mol. wt of the polypeptide was assessed to be 7707 by electrospray mass spectroscopy. Toxicity tests showed that the polypeptide is potent in crickets. By the DABITCjPlTC manual method, the sequence of four residues from the N-terminal of the polypeptide was determined to be Asp_,-Asn-Gly-TyrrJ, which is the same as that of BmK AS. Likewise, electrophysiological recording showed that the polypeptide also increases Na+ permeability on NGlOS-IS cells. Further sequence analysis of the first I6 N-terminal residues (except for two residues at positions 13 and.15 which are undefined) showed that two residues at positions Asp-7 and Val-14 in BmK AS are replaced by Asn-7 and Asn-14 in the purified polypeptide. Thus the purified polypeptide is considered to be a new analogue of BmK AS from the same venom. Scan qfsensitivities of Chinese scorpion venom of nine kinds of K+ or Cl- channels. Y. Liu,’ Y.-H. Ji.’ S. Oiki,’ A. Hazama,’ S. Morishima? R. Sabirov,’ S.-S. Azhou,? S. Hayashi,’ T. Tsumura,: T. Ito,? K. Furuya’ and Y. Okada’ (‘Shanghai Institute of Physiology, Shanghai 200031, P. R. China; and ‘National institute for Physiological Sciences, Okazaki 444, Japan). To date, eight K+ channel-blocking ligands and one Cl- channel-blocking ligand have been reported from the venoms of various scorpions. These ligands, composed of 31-39 amino acids, have served as useful tools for elucidating the function of kinds of relative K+ channels (Ji and Li, 1995). as well as promising pharmaceuticals, such as for the treatment of Parkinson’s disease. In this communication, the sensitivities of the venom and a minipeptide fraction of the Chinese scorpion, Buthus marrensi Karsch, on nine kinds of K+ or Cl- channels were primary examined, respectively. The results showed that, under voltage-clamp and patch-clamp cell-attached or whole-cell or inside-out recording conditions, of the nine channels tested, seven (inward rectified K+ channels expressed in Xenopus-oocytes cells, Ca2+-activated K’ channel and ATP-activated K+ channel of p-cells isolated from rat pancreas, CAMP-activated Cll channel in guinea-pig Paneth’s cells. CAMP-activated Cll channel in guinea-pig ventricular cells, and volume-sensitive Cl- channel in human small intestinal epithelial cell line T84) were completely insensitive, while a remarkable blocking effect on transient outward K+ channels was recorded in neonatal rat ventricular cells at 10.5 mg of the minipeptide fraction/ml concentration. The blocking effect was reversible after washing for 15 min. Likewise, it was recorded that the venom at 2.5 pg/ml concentration decreased the K’ current on voltage-gated K+ channels of NGlOS-15 cells. Ji, Y. H. and Liu, Y. (1995) Life Sci. 7, l-5,
10.
Functional multivalence and structural uniqueness of a new polypeptide .from Chinese scorpion Buthus martensi Kursch. Y. Liu.’ H.-Y. Huang,’ Q. Hu,’ H.-M. Ren,’ J. Zhao,’ B.-L. Song,’ C.-W. Zhou,’ J.-W. Zhang,’ Y.-L. Shi,’ Y.-H. Ji,’ T. Ohishi,* T. Mochizuki,’ M. Hashino’ and N. Yanaihara’ (‘Shanghai Institute of Physiology, Shanghai 2003 I. P.R. China; and ‘School of Pharmacological Science, University of Shizuoka, 52-l Yada. Shizuoka 422, Japan). Scorpion neurotoxins are well known to be useful probes for elucidating the function of kinds of relative ion channels. In this communication, we report the amino acid sequence and functional characteristics of a new polypeptide (BmK AS), composed of 63 amino acids from the Chinese scorpion Buthus martensi Karsch. By repeated high-performance liquid chromatography (HPLC), an active polypeptide BmK AS was purified and finally examined as a band in slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. The mol. wt of BmK AS was precisely determined to be 7696 by electrospray mass spectroscopy. The previous work mentioned that BmK AS seems to be a potent activator of ryanodine receptor on rabbit skeletal muscle (Ji et al.. 1996). Nevertheless, further pharmacological and electrophysiological experiments showed that this bioactive macromolecule remarkably affects [‘Hlnoradrenaline release on rat hippocampal slices and also enhances Na+ conductance on voltage-gated Na+ channels of NGlOS-15 cells. After the purified BmK AS polypeptide was reduced and S-carboxymethylated. as well as digested, respectively, with trypsin and S. aureus Vx, the complete amino acid sequence of BmK AS was established by sequence analysis of the native and modified BmK AS and the essential enzymatic fragments. The molecular structure of BmK AS showed poor similarity to known Na+ channel-blocking ligands from the same venom, but about 80% homology with that of AaH IT,, an anti-insect neurotoxin from the venom of African scorpion A. austrafis Hector, which has been described as a distinct Na+ channel-blocking ligand in both insects and mammals. Ji. Y. H. 6’1 al. (1996) Chin. Sci. Bull. (to be published). Pur$cation and sequence derermination qf a ne\r neutral mammalian neurotoxin from scorpion Buthus martensi Karsch. M. Luo.’ Y. Xiong,’ M. Wang,’ D. Wang’ and C. Chi’ (‘Shanghai Institute of Biochemistry, Academia
493
BmK AS, a bioactive polypeptide composed of 63 amino acids purified from the venom of Chinese scorpion Bufhus martensi Karsch (BmK) has been demonstrated to be a potent activator of the ryanodine receptor, and also enhances noradrenaline release and Na+ conductance. Here we report another BmK AS-like polypeptide from the same venom. The BmK venom was isolated by a series of courses, including gel filtration on Sephadex G-25, Sephadex G-50 columns and anion-exchange chromatography on a DEAE-Sephadex A-50 column. Finally, a new polypeptide was purified by repeated high-performance liquid chromatography (HPLC) and examined as a band in slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. The retention time of the purified polypeptide was quite different to that of the known BmK AS in the HPLC pattern. The mol. wt of the polypeptide was assessed to be 7707 by electrospray mass spectroscopy. Toxicity tests showed that the polypeptide is potent in crickets. By the DABITCjPlTC manual method, the sequence of four residues from the N-terminal of the polypeptide was determined to be Asp_,-Asn-Gly-TyrrJ, which is the same as that of BmK AS. Likewise, electrophysiological recording showed that the polypeptide also increases Na+ permeability on NGlOS-IS cells. Further sequence analysis of the first I6 N-terminal residues (except for two residues at positions 13 and.15 which are undefined) showed that two residues at positions Asp-7 and Val-14 in BmK AS are replaced by Asn-7 and Asn-14 in the purified polypeptide. Thus the purified polypeptide is considered to be a new analogue of BmK AS from the same venom. Scan qfsensitivities of Chinese scorpion venom of nine kinds of K+ or Cl- channels. Y. Liu,’ Y.-H. Ji.’ S. Oiki,’ A. Hazama,’ S. Morishima? R. Sabirov,’ S.-S. Azhou,? S. Hayashi,’ T. Tsumura,: T. Ito,? K. Furuya’ and Y. Okada’ (‘Shanghai Institute of Physiology, Shanghai 200031, P. R. China; and ‘National institute for Physiological Sciences, Okazaki 444, Japan). To date, eight K+ channel-blocking ligands and one Cl- channel-blocking ligand have been reported from the venoms of various scorpions. These ligands, composed of 31-39 amino acids, have served as useful tools for elucidating the function of kinds of relative K+ channels (Ji and Li, 1995). as well as promising pharmaceuticals, such as for the treatment of Parkinson’s disease. In this communication, the sensitivities of the venom and a minipeptide fraction of the Chinese scorpion, Buthus marrensi Karsch, on nine kinds of K+ or Cl- channels were primary examined, respectively. The results showed that, under voltage-clamp and patch-clamp cell-attached or whole-cell or inside-out recording conditions, of the nine channels tested, seven (inward rectified K+ channels expressed in Xenopus-oocytes cells, Ca2+-activated K’ channel and ATP-activated K+ channel of p-cells isolated from rat pancreas, CAMP-activated Cll channel in guinea-pig Paneth’s cells. CAMP-activated Cll channel in guinea-pig ventricular cells, and volume-sensitive Cl- channel in human small intestinal epithelial cell line T84) were completely insensitive, while a remarkable blocking effect on transient outward K+ channels was recorded in neonatal rat ventricular cells at 10.5 mg of the minipeptide fraction/ml concentration. The blocking effect was reversible after washing for 15 min. Likewise, it was recorded that the venom at 2.5 pg/ml concentration decreased the K’ current on voltage-gated K+ channels of NGlOS-15 cells. Ji, Y. H. and Liu, Y. (1995) Life Sci. 7, l-5,
10.
Functional multivalence and structural uniqueness of a new polypeptide .from Chinese scorpion Buthus martensi Kursch. Y. Liu.’ H.-Y. Huang,’ Q. Hu,’ H.-M. Ren,’ J. Zhao,’ B.-L. Song,’ C.-W. Zhou,’ J.-W. Zhang,’ Y.-L. Shi,’ Y.-H. Ji,’ T. Ohishi,* T. Mochizuki,’ M. Hashino’ and N. Yanaihara’ (‘Shanghai Institute of Physiology, Shanghai 2003 I. P.R. China; and ‘School of Pharmacological Science, University of Shizuoka, 52-l Yada. Shizuoka 422, Japan). Scorpion neurotoxins are well known to be useful probes for elucidating the function of kinds of relative ion channels. In this communication, we report the amino acid sequence and functional characteristics of a new polypeptide (BmK AS), composed of 63 amino acids from the Chinese scorpion Buthus martensi Karsch. By repeated high-performance liquid chromatography (HPLC), an active polypeptide BmK AS was purified and finally examined as a band in slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. The mol. wt of BmK AS was precisely determined to be 7696 by electrospray mass spectroscopy. The previous work mentioned that BmK AS seems to be a potent activator of ryanodine receptor on rabbit skeletal muscle (Ji et al.. 1996). Nevertheless, further pharmacological and electrophysiological experiments showed that this bioactive macromolecule remarkably affects [‘Hlnoradrenaline release on rat hippocampal slices and also enhances Na+ conductance on voltage-gated Na+ channels of NGlOS-15 cells. After the purified BmK AS polypeptide was reduced and S-carboxymethylated. as well as digested, respectively, with trypsin and S. aureus Vx, the complete amino acid sequence of BmK AS was established by sequence analysis of the native and modified BmK AS and the essential enzymatic fragments. The molecular structure of BmK AS showed poor similarity to known Na+ channel-blocking ligands from the same venom, but about 80% homology with that of AaH IT,, an anti-insect neurotoxin from the venom of African scorpion A. austrafis Hector, which has been described as a distinct Na+ channel-blocking ligand in both insects and mammals. Ji. Y. H. 6’1 al. (1996) Chin. Sci. Bull. (to be published). Pur$cation and sequence derermination qf a ne\r neutral mammalian neurotoxin from scorpion Buthus martensi Karsch. M. Luo.’ Y. Xiong,’ M. Wang,’ D. Wang’ and C. Chi’ (‘Shanghai Institute of Biochemistry, Academia