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Binding of 125I-toxin, from the spitting cobra Naja mossambica mossambica, at frog neuromuscular junctions
682
Short Communications
should also replace the use of human volunteers since it is much more sensitive than the cutaaeous pinprick method. AcApeawledgenrents-The sufhora acknowledge the aid and asaiatanct: of Mr . Dwln Cutco of the Chesapeake Biological Laboratory, Solomono, Md ; Dr. PAUL ZUHICOFF of the Virginia Institute of Marine Stdena, Gloucester Point, Virginia ; Mr . J>~eY Heettu of Boca Raton, Florida; Dr. F.nwARn J. Motezrnoly of the University of Maryland School of Pharmacy ; RoHexT Mn l.r_x and HEtENa RvsnvstEnv of Baltimore, Md . and Dr. Rtcx.~an Fewcarr of Easters New Mexico University. These investigations were supported by grant is aidNo . ES01474 from the National Institutes of Health . R)3F8Rl3NCF.S Btrnxsrr, J. W. and CAtmrl, G. J. (1973) Purification of sea >xttle nematocyst toxins by gel diffusion. Toxicoa 11, 243. BtrnrlErr, J. W. and C.v.~nox, G. J. (1974a) The enzymatic content of the veaoms of the sea nettle and the Portuguese man-0'-war. Comp. Bloshsnt. Phystol. 47B, 815. BURNEIT, J. W. and Cet~t+ox, G. J. (19746) Sea nettle and man-o'-war venotna: a chemical comparison of their venoms and studies on the pathogeaeais of the atiag. J. invest. Der»r. G2, 372. Btrnlverr, J. W. and Cw<.rrnv, G. J. (1977) The chemistry and toxicology of some venomous pelagic coelenterates. Toxicoa 1S, 177. Btrnrlsrr, J. W., CALhON, G. J., Mt:»rR, H. and ICePLex, A. P. 11975) Mediatotx present in the nematocyst venoma of the sea nettle, sea wasp and Portuguese man-o'-war . Conrp. Btochem. Physiol. S1C,1S3. Btrnlve~rr, J. W. and Gotn~, W. M. (1971) Further atutüea on the purification of physiological actions of sea nettle toxin. Pros. Soc. exp. Blot. Med,138, 759. Cua~ox, G. J. sad Boxlverr, J. W. (1973) The purification of Portuguese man-o'-war nematocyst toxins by gel diffusion. Comp . Gea. Pharawcol. 4, 267. F.1~tE, J. F. and SHANAMAN, J. (1963) Bradykinin writhing : a method for measuring analgesia . Proc . Soc. exp. Blol . Med. 114, 680. Hersreen, B. W. (1965) Phylum : Coeleaterata . In : Poisonous sad Vatomous Aalmals of the World, Vol. I, p. 297. Washington, U.S. Government Printing O®ce : Washington, D.C. W~nneLL, W. J. (1956) A simple ultraviolet method for the determination of protein . l. Lab. clip . Med. 48, 311.
Toztca~, Vol. 16, pD . tia?r686. O inn Prou Ltd. 1978 . Piintsd In ßceat
Hrluin.
BINDING OF 1~I-TOXIN, FROM THE SPITTING COBRA NAJA MOSSAMBICA MOSSAMBICA, AT FROG NEUROMUSCULAR JUNCTIONS MARIE TBS3IER, JEAN-MARIE GRILLO
and
JEAN MAMBRINI
Laboratoiro de $iochimie, Faculté de Médecine, Secteur Nord, 13326 Marseille Cedex 3, France Laboratoire d~üstologie, Faculté de Médecine, Secteuu Nord, 13326 Marseille Cedex 3, France, and Laboratoire de Physiologie Générale, Faculté des Sciences, 94000 Créteil, France (Aoaepted joyprbJkatiwt 7 February 1978)
the venoms of Elapidae and Hydrophidae snakes many curariform toxins have been purified, which are known to compete with acetylcholine at the postsynatic receptor. Some of these toxins have been used after labelling with tritium or lzsj, in order to localize, by electron microscopic autoradiography, the cholinergic receptor sites of the vertebrate neuromuscular junction (PORTER et al., 1973a, 19736; FERTUCK and SALPETER, 1976) and FROM
Short Communications
683
the electric organ of L'leclropltorus elec~ricus (Bouttceots e~ al., 1972) . From the venom of Naja ntossa»rbica mossambica three proteins have been purified and found to belong to the "short" postsynaptic toxin group which is characterized by a peptide chain of 60 to 62 amino acid residues cross-linked by 4 disulfide bridges (ROCHAT et al., 1974) . The complete sequences of two of them, neurotoxins 1 and III, have been determined (GREGOtRE and ROCHAT, 1977). This paper describes the binding ofthe ~ zsI-neurotoxin I of Naja mossombica nrossambica at the neuromuscular junctions of a frog sartorius nerve-muscle preparation . Toxin I of Naja mossambica mossambica was labelled using a technique described earlier (IZOC1iAT et al., 1976, 1977) . Two batches of tzsI-toxin were used whose specific radioactivities were respectively 2385 and 2173 curies per mmole (1 "3 and I ~2 atom 12sí incorporation per mole of protein) . The labelled toxin solution used contained 4"5 Itg t zsI-toxin I per ml of a 0"05 M sodium phosphate buffer, pH 7"4, containing 0" 1 ~ (w(v) of serum albumin. Freshly dissected sartorius muscles from frog were soaked in Ringer's buffer . When the motor nerve was stimulated by mechanical means, the muscles contracted. The preparations were dipped for 20 min in a bath containing 1 ml of the ~zsI-toxin solution and 3"5 ml of Ringer's, i .e. the final concentration of the toxin was 1 "4 x 10-' M. The muscles were then rinsed in Ringer's buffer for 5 min (several changes of solution). When the muscles were again stimulated through the motor nerve, they no longer contracted. Using a binocular lens. fragments, less than 1 mm3 in size, were dissected from the edges of the muscles, at places where nervous fibers were located . These were fixed with 2"5°ó glutaraldehyde in a
án G
N .ç 0 ó~ v 0 P
s
0
0
Olstance,
~am
FIG. Z. DISIRlBU710N FREQUENCY OF D1SrANCEa FROA! ORAINS 70 AXONAI. ON ALTIDRADiOORANa.
61EAaURFD
Axonal membrane is positioned at the zero point on abrissa axis. Onecan see that 53 ~ of the grains are located inside a 1~26 ~trn width band on the postsynaptic side of the synaptic cleft. Measurements were made on 17,500 erilarganents, clans width : 031 fun.
68 4
Short Communications
018 M sodium phosphate buffer, pH 7~3 (2 hr), post fixed for 1 ~5 hr with 2jo osmium tetraoxide and finally embedded in Epon 812. Autoradiogam of ultrafine longitudinal sections of endplates were made using llford L,, emulsion (Ilford Ltd., Essex, U.K .). After exposure periods ranging from 6 to 20 days, the specimens were developed in microdol-X Kodak at 18°C and examined in a Philips EM 201S electron microscope . The distribution of grains on the autoradiogram was heterogeneous with maximum concentrations on neuromuscular junctions. Most of the grains are located at the top of the functional folds on the subneural apparatus (Fig. I ). These observations were complemented by a numerit~l study of the distribution of grains over a 7 ltm wide zone situated on both sides of the axonal membrane : the distance of each grain to the axonal membrane was measured on the autoradiograms of three distinct end plates . The results were pooled and distributed on a 25 class histogram (Fig, 2). This histogram confirms that the tzsl-toxin I binds selectively on the subneural membrane since the grain density is maximum in a narrow hand (I Etm wide) on the muscle side of the neuromuscular junction . Our results are in agrcement with those Of FERTUCK and SALPEI'ER (1976) who used a-bungarotoxin and the stcrnomastoYd muscle of mice. Toxin I of Naja mossambica ntossanhica is a curariform neurotoxin able to bind selectively on the cholinergic receptor site of vertebrate muscle. Accordingly we may assume that it can be used not only as a highly specific label but also considering its very high specific radioactivity (more than 2000 CiÍmole) . as a very sensitive label. Acknowledgenurrts-The authors thank Professors Mttuho~, RocttAr ând VrrRY for helpful discussions . The work was supported by the Direction des Recherdtea et Etudes Techniques, the Crntre National de la Recherche Scientifique (ERA 070617) and the Institut National de la Recherche M~;dicale (U . 38) . RRF~RHj~jCE$ IIouRaeols, J. P., RvrEIt, A., MENFZ, A., Fttouuat:ort', P., BaQtre'r, P. and Ctuirceux, J . P. (1972) Localization of the cholinergic raeptor protein in £lectrophorus electroplax by high resolution autoradiography . FEBS. Letts. 25, 127. FERTUCR, H . C. and SAt.p~R, M . M . (1976) Quantitation of functional and extrajunctional acetylcholine receptors by electron microscope sutoradiography after ~ =aha-bungarotoxin binding at mouse neuromuscular junctions. J. Cell. Biol . 69, 144 . GReooate, J . and ROCIlAr, H . (1977) Amino acid sequences of neurotoxine I and 111 of the £lapidee snake Nato srossambka ntossasrb~a . Ettr . J. Bioduet . 80, 283 . PoRrttR, C. W., BARruRn, E . A. and CIIItv, T . H . í1973b) The ultrastructural localization and gtlantiuttion of cholinergic receptors at the mouse motor endplato . J . ~tentbr. Blol. 14, 383 . PoRreR, C. W., Cxtu, T. H ., Wlecxowslu, J. and BARNARD, A . (1973a) Types and locations of cholinergic receptor-like molecules in muscles fibres. Nature. New. Biol . 214, 3 . RocllAr, H., GRttaonee, J., MARrnv-Motrror, N ., M~vASHe, M ., ICOPEYAN, C . and MIRANDA, F. (19741 Purification of animal neurotoxine : isolation and characterization o . three neurotoxins from the venom of Nala nigrlcollis stossambica Peters . FEBS. Letts . 42, 335 . RocllAr, H ., Tt:~R, M ., MIRANDA, F . and LISSIrztcY, S . (1976) Enzymatic radioiodination of animal toxins to very high specißc radioactivity. In : Anistal, Plaat and Microbial Toxins, p. 8l, (OFISAICA, A ., HAYASHI, K . Snd $AWAt, Y., Eds.) . New York : Plenum . RocalAr, H ., Tttt
FIG . I . AUTORADIOORAM OF THE ENl}PUTS REOION OBTAINED AFTER INCUBATION OF A FROO NERVE-MUSCLE PREPARATION WITH ~=SI-TOXIN I OF NgJO l1.OJJOAtL~CG l1lOJSOAfGICG.
One can see that gains are mainly located at the top of the jlmctional folds on the aubneluai apparatus.
Short Communications
should also replace the use of human volunteers since it is much more sensitive than the cutaaeous pinprick method. AcApeawledgenrents-The sufhora acknowledge the aid and asaiatanct: of Mr . Dwln Cutco of the Chesapeake Biological Laboratory, Solomono, Md ; Dr. PAUL ZUHICOFF of the Virginia Institute of Marine Stdena, Gloucester Point, Virginia ; Mr . J>~eY Heettu of Boca Raton, Florida; Dr. F.nwARn J. Motezrnoly of the University of Maryland School of Pharmacy ; RoHexT Mn l.r_x and HEtENa RvsnvstEnv of Baltimore, Md . and Dr. Rtcx.~an Fewcarr of Easters New Mexico University. These investigations were supported by grant is aidNo . ES01474 from the National Institutes of Health . R)3F8Rl3NCF.S Btrnxsrr, J. W. and CAtmrl, G. J. (1973) Purification of sea >xttle nematocyst toxins by gel diffusion. Toxicoa 11, 243. BtrnrlErr, J. W. and C.v.~nox, G. J. (1974a) The enzymatic content of the veaoms of the sea nettle and the Portuguese man-0'-war. Comp. Bloshsnt. Phystol. 47B, 815. BURNEIT, J. W. and Cet~t+ox, G. J. (19746) Sea nettle and man-o'-war venotna: a chemical comparison of their venoms and studies on the pathogeaeais of the atiag. J. invest. Der»r. G2, 372. Btrnlverr, J. W. and Cw<.rrnv, G. J. (1977) The chemistry and toxicology of some venomous pelagic coelenterates. Toxicoa 1S, 177. Btrnrlsrr, J. W., CALhON, G. J., Mt:»rR, H. and ICePLex, A. P. 11975) Mediatotx present in the nematocyst venoma of the sea nettle, sea wasp and Portuguese man-o'-war . Conrp. Btochem. Physiol. S1C,1S3. Btrnlve~rr, J. W. and Gotn~, W. M. (1971) Further atutüea on the purification of physiological actions of sea nettle toxin. Pros. Soc. exp. Blot. Med,138, 759. Cua~ox, G. J. sad Boxlverr, J. W. (1973) The purification of Portuguese man-o'-war nematocyst toxins by gel diffusion. Comp . Gea. Pharawcol. 4, 267. F.1~tE, J. F. and SHANAMAN, J. (1963) Bradykinin writhing : a method for measuring analgesia . Proc . Soc. exp. Blol . Med. 114, 680. Hersreen, B. W. (1965) Phylum : Coeleaterata . In : Poisonous sad Vatomous Aalmals of the World, Vol. I, p. 297. Washington, U.S. Government Printing O®ce : Washington, D.C. W~nneLL, W. J. (1956) A simple ultraviolet method for the determination of protein . l. Lab. clip . Med. 48, 311.
Toztca~, Vol. 16, pD . tia?r686. O inn Prou Ltd. 1978 . Piintsd In ßceat
Hrluin.
BINDING OF 1~I-TOXIN, FROM THE SPITTING COBRA NAJA MOSSAMBICA MOSSAMBICA, AT FROG NEUROMUSCULAR JUNCTIONS MARIE TBS3IER, JEAN-MARIE GRILLO
and
JEAN MAMBRINI
Laboratoiro de $iochimie, Faculté de Médecine, Secteur Nord, 13326 Marseille Cedex 3, France Laboratoire d~üstologie, Faculté de Médecine, Secteuu Nord, 13326 Marseille Cedex 3, France, and Laboratoire de Physiologie Générale, Faculté des Sciences, 94000 Créteil, France (Aoaepted joyprbJkatiwt 7 February 1978)
the venoms of Elapidae and Hydrophidae snakes many curariform toxins have been purified, which are known to compete with acetylcholine at the postsynatic receptor. Some of these toxins have been used after labelling with tritium or lzsj, in order to localize, by electron microscopic autoradiography, the cholinergic receptor sites of the vertebrate neuromuscular junction (PORTER et al., 1973a, 19736; FERTUCK and SALPETER, 1976) and FROM
Short Communications
683
the electric organ of L'leclropltorus elec~ricus (Bouttceots e~ al., 1972) . From the venom of Naja ntossa»rbica mossambica three proteins have been purified and found to belong to the "short" postsynaptic toxin group which is characterized by a peptide chain of 60 to 62 amino acid residues cross-linked by 4 disulfide bridges (ROCHAT et al., 1974) . The complete sequences of two of them, neurotoxins 1 and III, have been determined (GREGOtRE and ROCHAT, 1977). This paper describes the binding ofthe ~ zsI-neurotoxin I of Naja mossombica nrossambica at the neuromuscular junctions of a frog sartorius nerve-muscle preparation . Toxin I of Naja mossambica mossambica was labelled using a technique described earlier (IZOC1iAT et al., 1976, 1977) . Two batches of tzsI-toxin were used whose specific radioactivities were respectively 2385 and 2173 curies per mmole (1 "3 and I ~2 atom 12sí incorporation per mole of protein) . The labelled toxin solution used contained 4"5 Itg t zsI-toxin I per ml of a 0"05 M sodium phosphate buffer, pH 7"4, containing 0" 1 ~ (w(v) of serum albumin. Freshly dissected sartorius muscles from frog were soaked in Ringer's buffer . When the motor nerve was stimulated by mechanical means, the muscles contracted. The preparations were dipped for 20 min in a bath containing 1 ml of the ~zsI-toxin solution and 3"5 ml of Ringer's, i .e. the final concentration of the toxin was 1 "4 x 10-' M. The muscles were then rinsed in Ringer's buffer for 5 min (several changes of solution). When the muscles were again stimulated through the motor nerve, they no longer contracted. Using a binocular lens. fragments, less than 1 mm3 in size, were dissected from the edges of the muscles, at places where nervous fibers were located . These were fixed with 2"5°ó glutaraldehyde in a
án G
N .ç 0 ó~ v 0 P
s
0
0
Olstance,
~am
FIG. Z. DISIRlBU710N FREQUENCY OF D1SrANCEa FROA! ORAINS 70 AXONAI. ON ALTIDRADiOORANa.
61EAaURFD
Axonal membrane is positioned at the zero point on abrissa axis. Onecan see that 53 ~ of the grains are located inside a 1~26 ~trn width band on the postsynaptic side of the synaptic cleft. Measurements were made on 17,500 erilarganents, clans width : 031 fun.
68 4
Short Communications
018 M sodium phosphate buffer, pH 7~3 (2 hr), post fixed for 1 ~5 hr with 2jo osmium tetraoxide and finally embedded in Epon 812. Autoradiogam of ultrafine longitudinal sections of endplates were made using llford L,, emulsion (Ilford Ltd., Essex, U.K .). After exposure periods ranging from 6 to 20 days, the specimens were developed in microdol-X Kodak at 18°C and examined in a Philips EM 201S electron microscope . The distribution of grains on the autoradiogram was heterogeneous with maximum concentrations on neuromuscular junctions. Most of the grains are located at the top of the functional folds on the subneural apparatus (Fig. I ). These observations were complemented by a numerit~l study of the distribution of grains over a 7 ltm wide zone situated on both sides of the axonal membrane : the distance of each grain to the axonal membrane was measured on the autoradiograms of three distinct end plates . The results were pooled and distributed on a 25 class histogram (Fig, 2). This histogram confirms that the tzsl-toxin I binds selectively on the subneural membrane since the grain density is maximum in a narrow hand (I Etm wide) on the muscle side of the neuromuscular junction . Our results are in agrcement with those Of FERTUCK and SALPEI'ER (1976) who used a-bungarotoxin and the stcrnomastoYd muscle of mice. Toxin I of Naja mossambica ntossanhica is a curariform neurotoxin able to bind selectively on the cholinergic receptor site of vertebrate muscle. Accordingly we may assume that it can be used not only as a highly specific label but also considering its very high specific radioactivity (more than 2000 CiÍmole) . as a very sensitive label. Acknowledgenurrts-The authors thank Professors Mttuho~, RocttAr ând VrrRY for helpful discussions . The work was supported by the Direction des Recherdtea et Etudes Techniques, the Crntre National de la Recherche Scientifique (ERA 070617) and the Institut National de la Recherche M~;dicale (U . 38) . RRF~RHj~jCE$ IIouRaeols, J. P., RvrEIt, A., MENFZ, A., Fttouuat:ort', P., BaQtre'r, P. and Ctuirceux, J . P. (1972) Localization of the cholinergic raeptor protein in £lectrophorus electroplax by high resolution autoradiography . FEBS. Letts. 25, 127. FERTUCR, H . C. and SAt.p~R, M . M . (1976) Quantitation of functional and extrajunctional acetylcholine receptors by electron microscope sutoradiography after ~ =aha-bungarotoxin binding at mouse neuromuscular junctions. J. Cell. Biol . 69, 144 . GReooate, J . and ROCIlAr, H . (1977) Amino acid sequences of neurotoxine I and 111 of the £lapidee snake Nato srossambka ntossasrb~a . Ettr . J. Bioduet . 80, 283 . PoRrttR, C. W., BARruRn, E . A. and CIIItv, T . H . í1973b) The ultrastructural localization and gtlantiuttion of cholinergic receptors at the mouse motor endplato . J . ~tentbr. Blol. 14, 383 . PoRreR, C. W., Cxtu, T. H ., Wlecxowslu, J. and BARNARD, A . (1973a) Types and locations of cholinergic receptor-like molecules in muscles fibres. Nature. New. Biol . 214, 3 . RocllAr, H., GRttaonee, J., MARrnv-Motrror, N ., M~vASHe, M ., ICOPEYAN, C . and MIRANDA, F. (19741 Purification of animal neurotoxine : isolation and characterization o . three neurotoxins from the venom of Nala nigrlcollis stossambica Peters . FEBS. Letts . 42, 335 . RocllAr, H ., Tt:~R, M ., MIRANDA, F . and LISSIrztcY, S . (1976) Enzymatic radioiodination of animal toxins to very high specißc radioactivity. In : Anistal, Plaat and Microbial Toxins, p. 8l, (OFISAICA, A ., HAYASHI, K . Snd $AWAt, Y., Eds.) . New York : Plenum . RocalAr, H ., Tttt
FIG . I . AUTORADIOORAM OF THE ENl}PUTS REOION OBTAINED AFTER INCUBATION OF A FROO NERVE-MUSCLE PREPARATION WITH ~=SI-TOXIN I OF NgJO l1.OJJOAtL~CG l1lOJSOAfGICG.
One can see that gains are mainly located at the top of the jlmctional folds on the aubneluai apparatus.