210
Biochimtca et Biophysica Acta, 1134(1992)210-21b © 1092ElsevierScience PublishersB.V. All rishtsresen,¢d0167.4889/92/$05.00
BBAMCR 13123
A n i o n i c currents o f chick sensory n e u r o n s are affected by a
phospholipase A 2 purified from the venom of the taipan snake L o u r i v a l D. P o s s a n i a, J a v i e r M o e h e a - M o r a l e s a, J o s e A m e z e u a a, B r i a n M. M a r t i n b, Gianfraneo Prestipino c and Mario Nobile c a Departamento de Bioqu[mica, Instituto de B,iotecnolog[a, UNAM, Cuernavaca (Mexico), b Molecular Neurogenetics Unit, Clinical Neuroscience Brancl~. National Institute of Mental Health, Bethesda, MD (USA) and c lstituto di Cibernetica e Bioflsica del CNR, Genova (Italy)
(Received 15 October 1991)
Key words: Dorsal root ganglion; Anionic channel; Whole-cell clamp; Phospholipase A2; Snake venom;
(Oxynraaus scuteUatus scutellatus)
A neurotoxic phospholipase A 2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A 2 (specific activity of 85 units/rag protein), and an apparent molecular weight of 16000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LDse ffi 10 ttg/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) 'dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na +, K + and Ca2+ currents, external application of phospholipase at a low concentration (30 aM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCI/in 140 mM CsCI), the current reversal potential revealed a shift in the positive direction typically due to CI- ion flux through the membrane. External application of a 50 ttM concentration of picrotoxin caused a reversible reduction of the phospholipaseinduced chloride current. Moreover, no appreciable current block was detected after addition of 50/tM DIDS.
Introduction Phospholipase A 2 (EC 3.1.1.4) seems to be an ubiquitous component of snake venoms [1,2]. It hydrolyses ester bonds from carbon inposition 2 of the glycerol moiety of phospholipids, and has been observed to cause myotoxic [3], cardiotoxic [4] and a variety of neurotoxic effects [5] in isolated tissues or experimental animals. Among the most neurotoxic pbospholipases are crotoxin from Cromlus durissus terrlfieus [6,7], ~8.bungaratoxin from Bungarus multicinctus [8,9], notexin from Notechis scutatus scutatus [10], taipoxin [11,12] and taicatoxin [13] from the taipan snake Oxyuranus scutellatus scutellatus. The action of these toxins are mainly at the neuromuscular junctions and affect neurotransmission by a direct action on the nerve
Correspondence: L.D. Possani, Departamento~ nioqulmica, lnstitaro de Biotecnologla, UNAM Apartadn Postal 510-3 Cuernavaca, Morelos 62271, Mexico.
terminal [1,14-76]. Two common characteristics of these toxins are the phospholipasic activity type A 2 and the association with one or more peptides, which make these neurotoxins a complex oligomeric structure, whose function is just started to be revealed. Specific neuronal binding sites have been described for /3-bungarotoxin [17], which is now known to block a subtype of voltage-dependent K + channel [18-20], taicatoxin which blocks Ca 2+ channels [13,21] and a phospholipase A 2 from Bungarus fasclatus, which affects calcium-modulated K + currents of epithelial cells [22]. Recently, a high affinity brain membrane binding site for a neurotoxic phospholipase purified from the taipan snake venom was reported [23]. Among the conclusions of the latter report [23] are a clear distinction between the catalytic activity of the phospholipase and the identification of a neuronal binding site in the synaptic membranes. These findings seem to support the idea that the catalytic activity for phospholipid hydroly_~is measured in vivo does not correlate with the lethality of these neurotoxic phospholipases [24,25].
211 Because the primary structure of the different phospholipases purified to date are similar, but not identical, it might be expected to find different affinities and/or specifities of these phosphelipases for their binding sites in excitable membranes. This communication reports the purification, chemical ci~aracterization and the effect of a newly purified phospholipase A 2 from the taipan snake venom on the anionic conductance, more likely chloride ck.annels, of chick dorsal root ganglion cells. An abstract containing part of this information was presented at 'IX Congress of Societa Italiana Biofisica Pura ed Aplicata', lsola D'Elba, Italy (May 12-18, 1990). Materials and Methods
Source of venom. Venom from the australian snake Oxyuranus scuteUatus scutellatus was obtained from the Laboratory 'La Nauyaca' (Emilio Carranza No. 487, M6xico, D.F. 09440, Mexico). Source of chemicals. All chemicals and solvents used were analytical reagents from companies indicated below. Water was deionized and double distilled over quartz. Picrotoxin and 4,4'-diisothiocyanostilhene-2,2'disulfonic acid (DIDS) were from Sigma (St. Louis, MI, USA). Purificationprocedures.Dried venom was sulubilized in 0.01 M Tris-Hcl buffer (pH 8.0) containing 0.01 M NaCI and centrifuged in a Sorvall centrifuge at 4°C, for 15 min using a rotor SS 34. Soluble venom was applied initially to a diethylaminoethyl-celluiose resin (DE-Cellulose, No. DE-32), prepared according to the specifications of the manufacturer (Whatman, Clifton, N J). This and subsequent columns were pre-equilibrated with appropriate buffers before loading the material. For dialysis the fractions were run against 1 I of the corresponding buffer for 15 rain with 4-5 changes. Dialysis tubing, Spectrapor Type 3 with a molecular weight cutoff of approx. 3500 (Spectrum Medical Industries, Los Angeles, CA) was used for dialysis. The material obtained from the first column was further separated by a cation exchanger resin of carboxymethyl-celluloso (CM-C.ellulose, No. CM-32, from Whatman) in 0.02 M ammonium acetate buffer (pH 4.7). The third step was a CM-ceHulose column equih'brated with 0.05 M phosphate buffer (pH 6.0). Elutiou from the CM-celluiose columns was performed with a continuous salt gradient of Nacl. The last step of purification was based on original data described by Rock and Snyder [26] using Sepharoso bound 1-(ll-carboxyundecyl)-2-hexadecyl-rac-glycero-3-phosphoryl-choline, here abbreviated PC-Sepharoso. Chemical characterization. Purity of sample was confirmed by polyacrylamide gel electrophoresis, either under non-denaturating conditions [27] or in the pres-
ence of sodium dodecyl sulfate [28], and by amino acid sequence determination of the N-terminal amino acid region of the enzyme by the Edman degradation procedure [29], using a Beckman 890 M microsequencer. Detailed information was previously published by our group [30]. Prior sequence determination the pure enzyme was reduced and alkylated as reported [30]. Amino acid composition was performed after hydrolysis in 6 M HCI [31] and derivatization with Edman reagent for high performance liquid chromatography (HPLC) of the carbamyl.derivatives [32]. Apparent molecular weight was determined from SDS gel electrophoresis [28] and continued by amino acid analysis. Lethality tests. The lethality of the various fractions was observed after intraperitoueal injection into mice (albino, CDI strain) of different amounts of protein in 0.1 to 0.2 ml isotonic solution containing a maximum of 50/~g protein, unless otherwise stated. A lethal dose 50% (LD50) was graphically determined by plotting the percentage of dead animals versus the logarithms of the phospholipase concentration, using six groups of four mice for each dose. It was assumed that 1 absorbancy unit at 280 nm in a cuvette of I cm pathlength equalled 1 mg/ml protein. Two designations were used to define toxicity of the various chromatographic components. 'Lethal' means that the toxic component at the dose injected killed the tested mouse within 20 h of injection. 'Nontoxic" means normal behavior similar to that observed following injection of 0.9% NaCI or injection of the buffers used during lmrification procedure.
Phospholipase activity and specO~eity. Phespholipase activity was measured by a fitrimetric method procedure with egg yolk as substrate [33], or by egg yolk embedded agaroso [34]. 1 unit of activity is defined as the amount of enzyme capable of releasing 1/tmol of unesterified fatty acid/rain, at 25°C (pH 8.0), with egg yolk emulsion as substrate. Determination of phespholipese specificity was performed by means of radio-labeled l ~ p h o l i p i d s (L-Idipalmitoyl-2(palmitoyl-l-t 4 ~ t i d ~ h o l i n e and l.-1-lysopalmitoyl-L-l[palmitoyl-1-t 4 ~ t i d y l c h o line) according to ~ and Van Roy [35]. Calcium dependence was determined by infu'bition of phospholipase activity in presence of EDTA (range from 0 to 50 raM), using a fixed amount o f ~ (2
nmol). Dorsal root gang//on c e k Thoracic and lumbar ganglia from 10- to 1 2 ~ chick embtyt~ were removed, treated and plated in cell culture medium, following essentially the same procedure described by Carbone and Lint [36]. E/ectmp~s/o/o~a/meamremen~ After 4 to 24 h plating the chick sensow neurons were used for lmtchclamp experiments [371. The various solutions used (see figure legends) contained tetraethylammonium (TEA),
212 cesium and cadmium as inhibitors of cation fluxes. The solutions were adjusted to pH 7.3 with NaOH, K O H or CsOH, depending on the experimental protocol and the osmolarity was adjusted to 290 m o s m o l / k g with mannitol. Aliquots of pure phospholipase, at the final concentrations desired, were added to the dishes or washed out from the dishes, according to the different experimental conditions described below. Whole-cell clamp currents evoked by the application of 200 ms long voltage steps from a holding potential (V h) of - 5 0 mV, were low-pass filtered at 3 kHz with a 8-pole Bessci filter, digitized at a sampling frequency of 10 kHz and stored on PDP 11/23 minicomputer for offline analysis [38]. All experiments were performed at room temperature (20 + 2°C).
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Biochimtca et Biophysica Acta, 1134(1992)210-21b © 1092ElsevierScience PublishersB.V. All rishtsresen,¢d0167.4889/92/$05.00
BBAMCR 13123
A n i o n i c currents o f chick sensory n e u r o n s are affected by a
phospholipase A 2 purified from the venom of the taipan snake L o u r i v a l D. P o s s a n i a, J a v i e r M o e h e a - M o r a l e s a, J o s e A m e z e u a a, B r i a n M. M a r t i n b, Gianfraneo Prestipino c and Mario Nobile c a Departamento de Bioqu[mica, Instituto de B,iotecnolog[a, UNAM, Cuernavaca (Mexico), b Molecular Neurogenetics Unit, Clinical Neuroscience Brancl~. National Institute of Mental Health, Bethesda, MD (USA) and c lstituto di Cibernetica e Bioflsica del CNR, Genova (Italy)
(Received 15 October 1991)
Key words: Dorsal root ganglion; Anionic channel; Whole-cell clamp; Phospholipase A2; Snake venom;
(Oxynraaus scuteUatus scutellatus)
A neurotoxic phospholipase A 2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A 2 (specific activity of 85 units/rag protein), and an apparent molecular weight of 16000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LDse ffi 10 ttg/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) 'dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na +, K + and Ca2+ currents, external application of phospholipase at a low concentration (30 aM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCI/in 140 mM CsCI), the current reversal potential revealed a shift in the positive direction typically due to CI- ion flux through the membrane. External application of a 50 ttM concentration of picrotoxin caused a reversible reduction of the phospholipaseinduced chloride current. Moreover, no appreciable current block was detected after addition of 50/tM DIDS.
Introduction Phospholipase A 2 (EC 3.1.1.4) seems to be an ubiquitous component of snake venoms [1,2]. It hydrolyses ester bonds from carbon inposition 2 of the glycerol moiety of phospholipids, and has been observed to cause myotoxic [3], cardiotoxic [4] and a variety of neurotoxic effects [5] in isolated tissues or experimental animals. Among the most neurotoxic pbospholipases are crotoxin from Cromlus durissus terrlfieus [6,7], ~8.bungaratoxin from Bungarus multicinctus [8,9], notexin from Notechis scutatus scutatus [10], taipoxin [11,12] and taicatoxin [13] from the taipan snake Oxyuranus scutellatus scutellatus. The action of these toxins are mainly at the neuromuscular junctions and affect neurotransmission by a direct action on the nerve
Correspondence: L.D. Possani, Departamento~ nioqulmica, lnstitaro de Biotecnologla, UNAM Apartadn Postal 510-3 Cuernavaca, Morelos 62271, Mexico.
terminal [1,14-76]. Two common characteristics of these toxins are the phospholipasic activity type A 2 and the association with one or more peptides, which make these neurotoxins a complex oligomeric structure, whose function is just started to be revealed. Specific neuronal binding sites have been described for /3-bungarotoxin [17], which is now known to block a subtype of voltage-dependent K + channel [18-20], taicatoxin which blocks Ca 2+ channels [13,21] and a phospholipase A 2 from Bungarus fasclatus, which affects calcium-modulated K + currents of epithelial cells [22]. Recently, a high affinity brain membrane binding site for a neurotoxic phospholipase purified from the taipan snake venom was reported [23]. Among the conclusions of the latter report [23] are a clear distinction between the catalytic activity of the phospholipase and the identification of a neuronal binding site in the synaptic membranes. These findings seem to support the idea that the catalytic activity for phospholipid hydroly_~is measured in vivo does not correlate with the lethality of these neurotoxic phospholipases [24,25].
211 Because the primary structure of the different phospholipases purified to date are similar, but not identical, it might be expected to find different affinities and/or specifities of these phosphelipases for their binding sites in excitable membranes. This communication reports the purification, chemical ci~aracterization and the effect of a newly purified phospholipase A 2 from the taipan snake venom on the anionic conductance, more likely chloride ck.annels, of chick dorsal root ganglion cells. An abstract containing part of this information was presented at 'IX Congress of Societa Italiana Biofisica Pura ed Aplicata', lsola D'Elba, Italy (May 12-18, 1990). Materials and Methods
Source of venom. Venom from the australian snake Oxyuranus scuteUatus scutellatus was obtained from the Laboratory 'La Nauyaca' (Emilio Carranza No. 487, M6xico, D.F. 09440, Mexico). Source of chemicals. All chemicals and solvents used were analytical reagents from companies indicated below. Water was deionized and double distilled over quartz. Picrotoxin and 4,4'-diisothiocyanostilhene-2,2'disulfonic acid (DIDS) were from Sigma (St. Louis, MI, USA). Purificationprocedures.Dried venom was sulubilized in 0.01 M Tris-Hcl buffer (pH 8.0) containing 0.01 M NaCI and centrifuged in a Sorvall centrifuge at 4°C, for 15 min using a rotor SS 34. Soluble venom was applied initially to a diethylaminoethyl-celluiose resin (DE-Cellulose, No. DE-32), prepared according to the specifications of the manufacturer (Whatman, Clifton, N J). This and subsequent columns were pre-equilibrated with appropriate buffers before loading the material. For dialysis the fractions were run against 1 I of the corresponding buffer for 15 rain with 4-5 changes. Dialysis tubing, Spectrapor Type 3 with a molecular weight cutoff of approx. 3500 (Spectrum Medical Industries, Los Angeles, CA) was used for dialysis. The material obtained from the first column was further separated by a cation exchanger resin of carboxymethyl-celluloso (CM-C.ellulose, No. CM-32, from Whatman) in 0.02 M ammonium acetate buffer (pH 4.7). The third step was a CM-ceHulose column equih'brated with 0.05 M phosphate buffer (pH 6.0). Elutiou from the CM-celluiose columns was performed with a continuous salt gradient of Nacl. The last step of purification was based on original data described by Rock and Snyder [26] using Sepharoso bound 1-(ll-carboxyundecyl)-2-hexadecyl-rac-glycero-3-phosphoryl-choline, here abbreviated PC-Sepharoso. Chemical characterization. Purity of sample was confirmed by polyacrylamide gel electrophoresis, either under non-denaturating conditions [27] or in the pres-
ence of sodium dodecyl sulfate [28], and by amino acid sequence determination of the N-terminal amino acid region of the enzyme by the Edman degradation procedure [29], using a Beckman 890 M microsequencer. Detailed information was previously published by our group [30]. Prior sequence determination the pure enzyme was reduced and alkylated as reported [30]. Amino acid composition was performed after hydrolysis in 6 M HCI [31] and derivatization with Edman reagent for high performance liquid chromatography (HPLC) of the carbamyl.derivatives [32]. Apparent molecular weight was determined from SDS gel electrophoresis [28] and continued by amino acid analysis. Lethality tests. The lethality of the various fractions was observed after intraperitoueal injection into mice (albino, CDI strain) of different amounts of protein in 0.1 to 0.2 ml isotonic solution containing a maximum of 50/~g protein, unless otherwise stated. A lethal dose 50% (LD50) was graphically determined by plotting the percentage of dead animals versus the logarithms of the phospholipase concentration, using six groups of four mice for each dose. It was assumed that 1 absorbancy unit at 280 nm in a cuvette of I cm pathlength equalled 1 mg/ml protein. Two designations were used to define toxicity of the various chromatographic components. 'Lethal' means that the toxic component at the dose injected killed the tested mouse within 20 h of injection. 'Nontoxic" means normal behavior similar to that observed following injection of 0.9% NaCI or injection of the buffers used during lmrification procedure.
Phospholipase activity and specO~eity. Phespholipase activity was measured by a fitrimetric method procedure with egg yolk as substrate [33], or by egg yolk embedded agaroso [34]. 1 unit of activity is defined as the amount of enzyme capable of releasing 1/tmol of unesterified fatty acid/rain, at 25°C (pH 8.0), with egg yolk emulsion as substrate. Determination of phespholipese specificity was performed by means of radio-labeled l ~ p h o l i p i d s (L-Idipalmitoyl-2(palmitoyl-l-t 4 ~ t i d ~ h o l i n e and l.-1-lysopalmitoyl-L-l[palmitoyl-1-t 4 ~ t i d y l c h o line) according to ~ and Van Roy [35]. Calcium dependence was determined by infu'bition of phospholipase activity in presence of EDTA (range from 0 to 50 raM), using a fixed amount o f ~ (2
nmol). Dorsal root gang//on c e k Thoracic and lumbar ganglia from 10- to 1 2 ~ chick embtyt~ were removed, treated and plated in cell culture medium, following essentially the same procedure described by Carbone and Lint [36]. E/ectmp~s/o/o~a/meamremen~ After 4 to 24 h plating the chick sensow neurons were used for lmtchclamp experiments [371. The various solutions used (see figure legends) contained tetraethylammonium (TEA),
212 cesium and cadmium as inhibitors of cation fluxes. The solutions were adjusted to pH 7.3 with NaOH, K O H or CsOH, depending on the experimental protocol and the osmolarity was adjusted to 290 m o s m o l / k g with mannitol. Aliquots of pure phospholipase, at the final concentrations desired, were added to the dishes or washed out from the dishes, according to the different experimental conditions described below. Whole-cell clamp currents evoked by the application of 200 ms long voltage steps from a holding potential (V h) of - 5 0 mV, were low-pass filtered at 3 kHz with a 8-pole Bessci filter, digitized at a sampling frequency of 10 kHz and stored on PDP 11/23 minicomputer for offline analysis [38]. All experiments were performed at room temperature (20 + 2°C).
lS--
•.
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j~'~"
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t ' " ' ~ r ,~e'-----L . . . . . . .
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