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Kinetics of ATP-release from liposomes by Streptolysin-O
259
Abstracts and amplitude of miniature-endplate potentials were decreased . ß-Bungarotoxin, another specific presynaptic neurotoxin, altered this amplitude in a manner indicating that these two toxins have distinct binding eaten but affect associated camponent(s) [1] . Saturable binding of active i 2 sl-BoNT to rat diaphragm preparations was demonstrated sutoradiographically at synaptic regions .
At central synapses, BoNT inhibited resting and R-stimulated release of acetylcholine from cerebrocortical synaptoeames ; also it decreased choline uptake to a limited extent . It failed to su~press Ca d + influx into synaptoiz somes . A saturable binding component for I-BoNT was demonstrated on synaptosomal membranes ; it was heatsenaitive and inactivated by trypsin. Its toxin affinity correlated with the concentrations of BoNT shown to affect transmitter release, possibly indicating some functional significance .
[1]
Dolly, J.O ., Gwilt, M., Tse, C .R . and FTray, D (1981) . J. PhyaioZ.
(In press)
Interaction between botulinum neurotoxin and ß-bungarotoxin at the rat neuromuscular junction . 32 .
KINETICS OF ATP-RELEASE FROM LIPOSOMES BY STREPTOLYSIN-0 . Hans HUSER, Christa-M. Schmidt and Franz-J . Fehrenbach Authors address : Department of Bacteriology, Robert Koch-Institut, Bundesgesundheitsamt Nordufer 20, 1000 Berlin 65, GFR. Interaction of C tol tic Toxins with artificial membranes have been studied wit ipi aspersions and liposomes (1-7) . Release of trapped mostly radioactive compounds from liposomes induced by cytolytic toxins, amphiphilic proteins and detergents have been measured . However, release kinetics of "marker" molecules could not be studied continously with the former techniques . The present work, therefore, presents kinetic data on the LO1 release of ATP from bounded vesicles by trepto ysinas measure with the bioluminescence metho Release kinetics of ATP from liposomes of total lipid extracts of sheep-, rabbit- and mouse - RBC reflected the well known species differences in RBC-sensitivity against SLO . ATP was released immediately after the addition of activated SLO . "tl/2-times" of ATP release were influenced by a change in cholesterol or phospholipid composition . ATP containing liposomes of defined constituents were stable for 4 month at 4o C . They can be used to standardize and compare toxin preparations of different sources . The kinetic assay was linear within a range of SLO concentration fron 1 .4 - 28 HU/ml at a liposome concentration equivalent to 5,4 nM of phospholipid .
26 0
Abstracts
References : ALOUF, J .E . and GEOFFROY, C . : FEMS Microbiology Letters 6 (1979) 413-416 . (7) BERNHEIMER, A.W ., COWELL, J .L . and KIM, K-S . : Biochim .Biophy .Acta 507 (19781 230-241 . (2) BERNHEIMER, A .W ., LINDER, R . and AVIGAD, L .S . : Infect .Immun . 23 (1979) 838-844 . (6) DUNCAN, J .L . and SCHLEGEL, A . : J .Cell Biol . 67 (1975) 160-173 . (1) FEHRENBACH, F .J . and EIBL, H. : Z .Naturforsch . 32c (1977) 101-109 . (3) FEHRENBACH,F .J ., RUSER, H . and Jaschinski, C . : FEMS Microbiology Letters 7 (1980) 285-288 . (8) JOHNSON, M .K ., GEOFFROY, C . and ALOUF,J .E . : Infect .Immun . 27 (1980) 97-101 . (5) PRIGENT, D . and ALOUF,J .E . : Labo-Pharma-Problêmes et Techniques 281 (1978) 910-913 . (4)
+ Poster presentation . STUDY OF MEMBRANE-BACTERIAL CYTOLYSINS INTERACTION USING A PHOTOREACTIVE
PROBE AND CHAOTROPIC AGENTS
Colette JOLIVET-REYNAUD I , Christiane GEOFFROY l , Jean IGOLEN 2 and Joseph E . ALOUF I
Unité des Antigènes Bactériens l (Equipe de Recherche Associée au CNRS N° and Unité de Chimie Or anique 2 Institut Pasteur, 28 rue du Docteur-Roux, 7574 Paris Cedex 15, France .
794)
The ql coli id 12- 4-azi do-2-nitrophenoxy)-steroyl-[1- 14C]-glucosamine (12-A - c is mown to nsert spontaneously nto the membrane bilaver. It sélect vely labels intrinsic membrane proteins (3) . The photoreactive group of this probe is located on the stearic acid chain and reacts with vicinal compounds only when sales are exposed to UV light . Such a probe has been used for studying membrane penetration by cholera toxin subunits in Newcastle disease virus membrane (4) .
In this communication, we describe the use of 12-APS-G1cN for the study of the interaction with sheep erythrocytes of the sulfhydryl-dependent toxin alveolysin (2) from aacillus alvei and clostrtat~ perfringens delta-toxin (1) . These two protein toxins which lyse eukaryotic cells are functionnally and chemically unrelated. We also describe the effects of chaotropic ions on toxin extractability after interaction with erythrocytes . Slab SDS-PAGE analysis of membrane erythrocytes constituents following toxin-induced cytolysis showed toxin association to membrane at free toxin Rf . Autoradio4rams of SDS-PAGE of membranes obtained after lvsis by alveolvsin and ae tT~a-torn respectivelyand probing with 12-APS-G1cN showed that only alveo-
Abstracts and amplitude of miniature-endplate potentials were decreased . ß-Bungarotoxin, another specific presynaptic neurotoxin, altered this amplitude in a manner indicating that these two toxins have distinct binding eaten but affect associated camponent(s) [1] . Saturable binding of active i 2 sl-BoNT to rat diaphragm preparations was demonstrated sutoradiographically at synaptic regions .
At central synapses, BoNT inhibited resting and R-stimulated release of acetylcholine from cerebrocortical synaptoeames ; also it decreased choline uptake to a limited extent . It failed to su~press Ca d + influx into synaptoiz somes . A saturable binding component for I-BoNT was demonstrated on synaptosomal membranes ; it was heatsenaitive and inactivated by trypsin. Its toxin affinity correlated with the concentrations of BoNT shown to affect transmitter release, possibly indicating some functional significance .
[1]
Dolly, J.O ., Gwilt, M., Tse, C .R . and FTray, D (1981) . J. PhyaioZ.
(In press)
Interaction between botulinum neurotoxin and ß-bungarotoxin at the rat neuromuscular junction . 32 .
KINETICS OF ATP-RELEASE FROM LIPOSOMES BY STREPTOLYSIN-0 . Hans HUSER, Christa-M. Schmidt and Franz-J . Fehrenbach Authors address : Department of Bacteriology, Robert Koch-Institut, Bundesgesundheitsamt Nordufer 20, 1000 Berlin 65, GFR. Interaction of C tol tic Toxins with artificial membranes have been studied wit ipi aspersions and liposomes (1-7) . Release of trapped mostly radioactive compounds from liposomes induced by cytolytic toxins, amphiphilic proteins and detergents have been measured . However, release kinetics of "marker" molecules could not be studied continously with the former techniques . The present work, therefore, presents kinetic data on the LO1 release of ATP from bounded vesicles by trepto ysinas measure with the bioluminescence metho Release kinetics of ATP from liposomes of total lipid extracts of sheep-, rabbit- and mouse - RBC reflected the well known species differences in RBC-sensitivity against SLO . ATP was released immediately after the addition of activated SLO . "tl/2-times" of ATP release were influenced by a change in cholesterol or phospholipid composition . ATP containing liposomes of defined constituents were stable for 4 month at 4o C . They can be used to standardize and compare toxin preparations of different sources . The kinetic assay was linear within a range of SLO concentration fron 1 .4 - 28 HU/ml at a liposome concentration equivalent to 5,4 nM of phospholipid .
26 0
Abstracts
References : ALOUF, J .E . and GEOFFROY, C . : FEMS Microbiology Letters 6 (1979) 413-416 . (7) BERNHEIMER, A.W ., COWELL, J .L . and KIM, K-S . : Biochim .Biophy .Acta 507 (19781 230-241 . (2) BERNHEIMER, A .W ., LINDER, R . and AVIGAD, L .S . : Infect .Immun . 23 (1979) 838-844 . (6) DUNCAN, J .L . and SCHLEGEL, A . : J .Cell Biol . 67 (1975) 160-173 . (1) FEHRENBACH, F .J . and EIBL, H. : Z .Naturforsch . 32c (1977) 101-109 . (3) FEHRENBACH,F .J ., RUSER, H . and Jaschinski, C . : FEMS Microbiology Letters 7 (1980) 285-288 . (8) JOHNSON, M .K ., GEOFFROY, C . and ALOUF,J .E . : Infect .Immun . 27 (1980) 97-101 . (5) PRIGENT, D . and ALOUF,J .E . : Labo-Pharma-Problêmes et Techniques 281 (1978) 910-913 . (4)
+ Poster presentation . STUDY OF MEMBRANE-BACTERIAL CYTOLYSINS INTERACTION USING A PHOTOREACTIVE
PROBE AND CHAOTROPIC AGENTS
Colette JOLIVET-REYNAUD I , Christiane GEOFFROY l , Jean IGOLEN 2 and Joseph E . ALOUF I
Unité des Antigènes Bactériens l (Equipe de Recherche Associée au CNRS N° and Unité de Chimie Or anique 2 Institut Pasteur, 28 rue du Docteur-Roux, 7574 Paris Cedex 15, France .
794)
The ql coli id 12- 4-azi do-2-nitrophenoxy)-steroyl-[1- 14C]-glucosamine (12-A - c is mown to nsert spontaneously nto the membrane bilaver. It sélect vely labels intrinsic membrane proteins (3) . The photoreactive group of this probe is located on the stearic acid chain and reacts with vicinal compounds only when sales are exposed to UV light . Such a probe has been used for studying membrane penetration by cholera toxin subunits in Newcastle disease virus membrane (4) .
In this communication, we describe the use of 12-APS-G1cN for the study of the interaction with sheep erythrocytes of the sulfhydryl-dependent toxin alveolysin (2) from aacillus alvei and clostrtat~ perfringens delta-toxin (1) . These two protein toxins which lyse eukaryotic cells are functionnally and chemically unrelated. We also describe the effects of chaotropic ions on toxin extractability after interaction with erythrocytes . Slab SDS-PAGE analysis of membrane erythrocytes constituents following toxin-induced cytolysis showed toxin association to membrane at free toxin Rf . Autoradio4rams of SDS-PAGE of membranes obtained after lvsis by alveolvsin and ae tT~a-torn respectivelyand probing with 12-APS-G1cN showed that only alveo-