NOTES
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 10/23/14 For personal use only.
Acknowledgements K.J.B. was supported by the Indiana University School of Medicine as part of the Student Research Program in Academic Medicine. Helpful discussions with Drs. Wei Liu and Beat U. Raess are appreciated. Birchrneier, W., and Christen, P. 1974. The reaction of cytoplasmic aspartate aminotransferase with bromopyruvate. J. Biol. Chem. 249: 6311-6315. Bowery, N.G., and Nistico, G. 1989. GABA: basic research and clinical applications. Pythagora Press. Rome-Milan. Der Garabedian P.A., Lotti, A.-M., and Vermeersch, J.J. 1986. 4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Eur. J. Biochem. 156: 589-596. Fonda, M.L. 1976. Bromopyruvate inactivation of glutamate apodecarboxylase. J. Biol. Chem. 251: 229-235. Hearl, W.G., and Churchich, J.E. 1984. Interactions between 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, two rnitochondrial enzymes. J. Biol. Chem. 259: 11 459 - 11 463. Kim, D.S., and Churchich, J.E. 1987. The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. J. Biol. Chem. 262: 14 250 - 14 254. KO, Y.H., and McFadden, B.A. 1990. Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. Arch. Biochem. Biophys. 278: 373-380. Liu, W., and Tsou, C.-L. 1986. Determination of rate constants for the irreversible inhibition of acetylcholine esterase by continuously monitoring the substrate reaction in the presence of the inhibitor. Biochim. Biophys. Acta, 870: 185-190.
Lowe, P.N., and Perham, R.N. 1984. Bromopyruvate as an activesite-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli. Biochemistry, 23: 91-97. Okamoto, M., and Morino, Y. 1973. Affinity labeling of aspartate aminotransferase isozymes by bromopyruvate. J. Biol. Chem. 248: 82-90. Rogawski, M.A., and Porter, R. J. 1990. Antiepileptic drugs: pharmacological mechanisms and clinical efficacy with consideration of promising developmental stage compounds. Pharmacol. Rev. 42: 223-286. Scott, E.M., and Jacoby, W.B. 1959. Soluble Y-aminobutyricglutamic-transaminase from Pseudomonasfluorescens.J. Biol. Chem. 234: 932-936. Tian, W.-X., and Tsou, C.-L. 1982. Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier. Biochemistry, 21: 1028-1032. Tsou, C.-L. 1988. Kinetics of substrate reactions during irreversible modification of enzyme activity. Adv. Enzymol. 61: 381436. Tunnicliff, G. 1986. GABA transaminase. In Neuromethods. Vol. 5. Edited by A.A. Boulton, G.B. Baker, and P.H. Yu. Humana Press, Clifton. pp. 389-419. Tunnicliff, G. 1991. GABA aminotransferase inhibitors as potential antiepileptic agents. In GABA mechanisms in epilepsy. Edited by G. Tunnicliff and B.U. Raess. Wiley-Liss, New York. pp. 189-204. Tunnicliff, G., and Ngo, T.T. 1978. Mechanism of inactivation of brain glutamic decarboxylase by 3-bromopyruvate. Int. J. Biochem. 9: 249-252. Yonaha, K., Suzuki, K., and Toyama, S. 1985.4-Aminobutyrate: 2-oxoglutarate of Streptomyces griseus. Eur. J. Biochem. 146: 101-106.
Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus CLAUDE GICQUAUD AND MICHELPARE Universite du Quebec h Trois-RiviPres, C.P. 500, Trois-RiviPres (Quebec), Canada G9A 5H7 Received December 16. 1991 GICQUAUD, C., and PARE,M. 1992. Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus. Biochem. Cell Biol. 70: 719-723. Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoebaprotacs cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin. Key words: phalloidin, virotoxin, motility, actin, membrane. GICQUAUD, C., et PARE,M. 1992. Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus. Biochem. Cell Biol. 70 : 7 19-723. Les virotoxines et la phalloidine sont des peptides qui induisent la polymtrisation de I'actine in vitro. Nous avons compart I'effet de cinq virotoxines et de la phalloydine sur I'ultrastmcture de prtparations ttaltes du cytoplasme d'Amoeba proteus. Comme la phallo'idine, les cinq virotoxines induisent la polymkrisation de I'actine cytoplasmique. De plus, les virotoxines, mais non la phalloidine, induisent la fragmentation des membranes en de petites vtsicules sphtriques. Nous concluons donc qu'en plus de l'actine, ces virotoxines peuvent agir sur une autre cible lite a la membrane. Mots cles : phalloidine, virotoxine, motilitk, actine, membrane. [Traduit par la rtdaction] ABBREVIATION: HPLC, high performance liquid chromatography. Printed in Canada / Imprime au Canada
719
BIOCHEM. CELL BIOL. VOL. 70,
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Introduction Virotoxins are a group of cyclic heptapeptides that are extracted from the deadly mushroom Amanita virosa (Faulstich et al. 1980, 1981; Thelestam and Gross 1990; Wieland 1986). We have previously established that these compounds have the remarkable property to bind specifically t o actin, inducing its polymerization into filaments. The resulting polymer is more resistant to proteases, heat denaturation, cytochalasin B, DNAse I, and chaotropic ions (Gicquaud et al. 1982). It would then appear that virotoxins are very similar to phalloidin with respect to their effects on actin polymerization in vitro. Their chemical structures, however, differ significantly. Indeed, contrary to phalloidin, virotoxins are monocyclic and contain the rare amino acids methyl sulfonyl tryptophan and dihydroxyproline (Table 1) (Faulstich et al. 1980). Very few studies have focused on the effects of virotoxins on cells or cell preparations. This is partly due to the fact that the cell membrane is not permeable to virotoxins. We have previously developed a technique that permits the mechanical removal of the cell membrane in a medium which maintains the integrity of the cell organelles and motility of the cytoplasm (Gicquaud and Couillard 1970, 1972). In these preparations, the cytoplasm is in direct contact with the medium. Using this technique, we have previously shown that phalloidin induces actin polymerization in the cytoplasm of Amoebaproteus, resulting in the inhibition of its motility (Paulin-Levasseur and Gicquaud 1981). In the present work, we have used the same approach and have found that, like phalloidin, the virotoxins polymerize actin and inhibit the motility of isolated Amoeba proteus cytoplasm. But also, a new important effect is reported: virotoxins fragment membranes, resulting in the formation of small vesicles. Thus, virotoxins, in addition to being able to interact with the actin molecule, have a second target located in the membrane. Materials and methods Cell culture Amoeba proteus (Pallas) was obtained as a clone established
from a strain from the General Biological Supply House (Turtox). It was cultured by the method of Prescott and Carrier (1964) and fed with Tetrahymena pyriformis. Prior to use, amoebae were fasted for 48 h to reduce the number of food vacuoles. Preparation of virotoxins and phalloidin
Virotoxins and phalloidin were extracted from the mushroom Amanita virosa harvested in the Parc national de la Mauricie, Que.
The mushrooms were kept as a lyophylized powder until extraction of peptides. Crude extraction of the toxins was carried out with methanolchloroform following the procedure of Yocum and Simons (1977). Phalloidin was separated from the virotoxins by chromatography on Sephadex LH20 as previously described (Gicquaud et al. 1982). Finally, the virotoxins were separated and phalloidin was purified by HPLC using a semipreparativecolumn (C18 Bondapak, 0.7 x 30 cm). Elution was performed with a linear gradient of 15-70% acetonitrile in water (Turcotte et al. 1984). Peptides were identified by HPLC chromatography using true standards provided by Dr. Faulstich and by amino acid analysis. Preparation of Amoeba proteus cytoplasm Preparation of demembraned cytoplasm was carried out as previously described (Gicquaud and Couillard 1970). Briefly, one hundred cells were washed in the incubation medium containing 1 mM ATP, 25 mM Tris-HC1, 0.75 mM EGTA, 2 mM MgCI,,
1992
TABLE1. Structure of virotoxins as determined by Faulstich et al. (1980) 7
6
-
CO-NH
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 10/23/14 For personal use only.
Acknowledgements K.J.B. was supported by the Indiana University School of Medicine as part of the Student Research Program in Academic Medicine. Helpful discussions with Drs. Wei Liu and Beat U. Raess are appreciated. Birchrneier, W., and Christen, P. 1974. The reaction of cytoplasmic aspartate aminotransferase with bromopyruvate. J. Biol. Chem. 249: 6311-6315. Bowery, N.G., and Nistico, G. 1989. GABA: basic research and clinical applications. Pythagora Press. Rome-Milan. Der Garabedian P.A., Lotti, A.-M., and Vermeersch, J.J. 1986. 4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Eur. J. Biochem. 156: 589-596. Fonda, M.L. 1976. Bromopyruvate inactivation of glutamate apodecarboxylase. J. Biol. Chem. 251: 229-235. Hearl, W.G., and Churchich, J.E. 1984. Interactions between 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, two rnitochondrial enzymes. J. Biol. Chem. 259: 11 459 - 11 463. Kim, D.S., and Churchich, J.E. 1987. The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. J. Biol. Chem. 262: 14 250 - 14 254. KO, Y.H., and McFadden, B.A. 1990. Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. Arch. Biochem. Biophys. 278: 373-380. Liu, W., and Tsou, C.-L. 1986. Determination of rate constants for the irreversible inhibition of acetylcholine esterase by continuously monitoring the substrate reaction in the presence of the inhibitor. Biochim. Biophys. Acta, 870: 185-190.
Lowe, P.N., and Perham, R.N. 1984. Bromopyruvate as an activesite-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli. Biochemistry, 23: 91-97. Okamoto, M., and Morino, Y. 1973. Affinity labeling of aspartate aminotransferase isozymes by bromopyruvate. J. Biol. Chem. 248: 82-90. Rogawski, M.A., and Porter, R. J. 1990. Antiepileptic drugs: pharmacological mechanisms and clinical efficacy with consideration of promising developmental stage compounds. Pharmacol. Rev. 42: 223-286. Scott, E.M., and Jacoby, W.B. 1959. Soluble Y-aminobutyricglutamic-transaminase from Pseudomonasfluorescens.J. Biol. Chem. 234: 932-936. Tian, W.-X., and Tsou, C.-L. 1982. Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier. Biochemistry, 21: 1028-1032. Tsou, C.-L. 1988. Kinetics of substrate reactions during irreversible modification of enzyme activity. Adv. Enzymol. 61: 381436. Tunnicliff, G. 1986. GABA transaminase. In Neuromethods. Vol. 5. Edited by A.A. Boulton, G.B. Baker, and P.H. Yu. Humana Press, Clifton. pp. 389-419. Tunnicliff, G. 1991. GABA aminotransferase inhibitors as potential antiepileptic agents. In GABA mechanisms in epilepsy. Edited by G. Tunnicliff and B.U. Raess. Wiley-Liss, New York. pp. 189-204. Tunnicliff, G., and Ngo, T.T. 1978. Mechanism of inactivation of brain glutamic decarboxylase by 3-bromopyruvate. Int. J. Biochem. 9: 249-252. Yonaha, K., Suzuki, K., and Toyama, S. 1985.4-Aminobutyrate: 2-oxoglutarate of Streptomyces griseus. Eur. J. Biochem. 146: 101-106.
Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus CLAUDE GICQUAUD AND MICHELPARE Universite du Quebec h Trois-RiviPres, C.P. 500, Trois-RiviPres (Quebec), Canada G9A 5H7 Received December 16. 1991 GICQUAUD, C., and PARE,M. 1992. Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus. Biochem. Cell Biol. 70: 719-723. Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoebaprotacs cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin. Key words: phalloidin, virotoxin, motility, actin, membrane. GICQUAUD, C., et PARE,M. 1992. Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus. Biochem. Cell Biol. 70 : 7 19-723. Les virotoxines et la phalloidine sont des peptides qui induisent la polymtrisation de I'actine in vitro. Nous avons compart I'effet de cinq virotoxines et de la phalloydine sur I'ultrastmcture de prtparations ttaltes du cytoplasme d'Amoeba proteus. Comme la phallo'idine, les cinq virotoxines induisent la polymkrisation de I'actine cytoplasmique. De plus, les virotoxines, mais non la phalloidine, induisent la fragmentation des membranes en de petites vtsicules sphtriques. Nous concluons donc qu'en plus de l'actine, ces virotoxines peuvent agir sur une autre cible lite a la membrane. Mots cles : phalloidine, virotoxine, motilitk, actine, membrane. [Traduit par la rtdaction] ABBREVIATION: HPLC, high performance liquid chromatography. Printed in Canada / Imprime au Canada
719
BIOCHEM. CELL BIOL. VOL. 70,
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 10/23/14 For personal use only.
720
Introduction Virotoxins are a group of cyclic heptapeptides that are extracted from the deadly mushroom Amanita virosa (Faulstich et al. 1980, 1981; Thelestam and Gross 1990; Wieland 1986). We have previously established that these compounds have the remarkable property to bind specifically t o actin, inducing its polymerization into filaments. The resulting polymer is more resistant to proteases, heat denaturation, cytochalasin B, DNAse I, and chaotropic ions (Gicquaud et al. 1982). It would then appear that virotoxins are very similar to phalloidin with respect to their effects on actin polymerization in vitro. Their chemical structures, however, differ significantly. Indeed, contrary to phalloidin, virotoxins are monocyclic and contain the rare amino acids methyl sulfonyl tryptophan and dihydroxyproline (Table 1) (Faulstich et al. 1980). Very few studies have focused on the effects of virotoxins on cells or cell preparations. This is partly due to the fact that the cell membrane is not permeable to virotoxins. We have previously developed a technique that permits the mechanical removal of the cell membrane in a medium which maintains the integrity of the cell organelles and motility of the cytoplasm (Gicquaud and Couillard 1970, 1972). In these preparations, the cytoplasm is in direct contact with the medium. Using this technique, we have previously shown that phalloidin induces actin polymerization in the cytoplasm of Amoebaproteus, resulting in the inhibition of its motility (Paulin-Levasseur and Gicquaud 1981). In the present work, we have used the same approach and have found that, like phalloidin, the virotoxins polymerize actin and inhibit the motility of isolated Amoeba proteus cytoplasm. But also, a new important effect is reported: virotoxins fragment membranes, resulting in the formation of small vesicles. Thus, virotoxins, in addition to being able to interact with the actin molecule, have a second target located in the membrane. Materials and methods Cell culture Amoeba proteus (Pallas) was obtained as a clone established
from a strain from the General Biological Supply House (Turtox). It was cultured by the method of Prescott and Carrier (1964) and fed with Tetrahymena pyriformis. Prior to use, amoebae were fasted for 48 h to reduce the number of food vacuoles. Preparation of virotoxins and phalloidin
Virotoxins and phalloidin were extracted from the mushroom Amanita virosa harvested in the Parc national de la Mauricie, Que.
The mushrooms were kept as a lyophylized powder until extraction of peptides. Crude extraction of the toxins was carried out with methanolchloroform following the procedure of Yocum and Simons (1977). Phalloidin was separated from the virotoxins by chromatography on Sephadex LH20 as previously described (Gicquaud et al. 1982). Finally, the virotoxins were separated and phalloidin was purified by HPLC using a semipreparativecolumn (C18 Bondapak, 0.7 x 30 cm). Elution was performed with a linear gradient of 15-70% acetonitrile in water (Turcotte et al. 1984). Peptides were identified by HPLC chromatography using true standards provided by Dr. Faulstich and by amino acid analysis. Preparation of Amoeba proteus cytoplasm Preparation of demembraned cytoplasm was carried out as previously described (Gicquaud and Couillard 1970). Briefly, one hundred cells were washed in the incubation medium containing 1 mM ATP, 25 mM Tris-HC1, 0.75 mM EGTA, 2 mM MgCI,,
1992
TABLE1. Structure of virotoxins as determined by Faulstich et al. (1980) 7
6
-
CO-NH
