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Botulinum neurotoxin as a probe for studying neurotransmitter release
258
Abatracta
tyrosyl residue per molecule located in fragment A and identified as tyrosine
60 after micro dansyl Edman degradation of the nitra-
ted tryptic peptide purified by ion exchange chromatography .
This
modification does not change the general conformation of the protein (as shown by immunochemical techniques and circular diohroism data) .Nitration of three non toxic cross-reacting forms of toxin resulting from missense mutation (crm 176,
crm 197 and crm 228)
are compared with that of the wild type protein . NAD or adenine protects diphtheria toxin from tyrosine 60 nitration but not the crm's .
Results suggest that tyrosine 60 is
implicated in the adenine binding site for the substrate NAD or for other nucleotides like ATP as proposed by Lory and Collier . Beugnier, N . Lory, S .
sad Zaaea,
J.
sad Collier, R .J .
(1977) Biochim .,Biophys . (1980)
Proc . Natl .
Acad .
Acta,490,225-234 Sci .
US A
77, 267-271 . Lory,
S .,
255,
Carroll, S .F .
and Collier, R .J .
(1980) J . Biol .
Chem .
12016-12019 . BOTULINUM NEUROTOSIN AS A PROBE FOR STUDYING NEUROTRANSMITTER RELEASE J .O . DOLLY I , C .K . TSE 1 , J . BLACRI , R . WILLIAMS I , P . HAMBLETON z and J . MELLING z 1 Department of Biochemistry, Imperial College, London and z C .A .M .R .~Porton Down, U .R .
It is known that botulinum toxin, produced by Clostridium botuZi~ttan, irreversibly and specifically inhibits release of acetylcholine from nerve terminals, with unique potency . Unavailability of adequate quantities of the pure neurotozin has forced moat investigators to use its high molecular weight complea(es) with haemagglutinin . Ia thin study, large amounts of neurotozin (Type A) were, therefore, purified to homogeneity and radiolabelled . Thin allowed it to be employed as a probe for nerve membrane components concerned with transmitter release at peripheral and central synapses .
Following isolation of the tozin complea by conventional methods, use of affinity and ion-ezchange chromatography ~ave a good yield (23% of that in the initial culture) of highly active (10 mouse LDsa/mg protein) and homogeneous neurotozin (BoNT) that was completely free of haemagglutinin . It gave a single sharp band on SDS polyacrylamide gel electrophoresis under non-reducing conditions, with a molecular weight of 140,000 ; after reduction equal amounts of two polypeptides (Mr of 99,000 and 55,000) were present . Likewise, one protein band was seen on isoelectric focussing with pI~6 .3 and in double immunodiffuaion gels using horse antibodies against the complex . izs l-Iodination of BoNT to high specific activity (>2,000Ci/mmol) was achieved with good retention of neuro-toxicity ; both subunits were labelled . In vitro application of BoNT to a frog neuromuscular preparation produced near-complete inhibition of transmission, without any detectable poatsynaptic effects . Injection of the BoNT into rat leg muscle produced paralysis ; the frequency
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 .
Abatracta
tyrosyl residue per molecule located in fragment A and identified as tyrosine
60 after micro dansyl Edman degradation of the nitra-
ted tryptic peptide purified by ion exchange chromatography .
This
modification does not change the general conformation of the protein (as shown by immunochemical techniques and circular diohroism data) .Nitration of three non toxic cross-reacting forms of toxin resulting from missense mutation (crm 176,
crm 197 and crm 228)
are compared with that of the wild type protein . NAD or adenine protects diphtheria toxin from tyrosine 60 nitration but not the crm's .
Results suggest that tyrosine 60 is
implicated in the adenine binding site for the substrate NAD or for other nucleotides like ATP as proposed by Lory and Collier . Beugnier, N . Lory, S .
sad Zaaea,
J.
sad Collier, R .J .
(1977) Biochim .,Biophys . (1980)
Proc . Natl .
Acad .
Acta,490,225-234 Sci .
US A
77, 267-271 . Lory,
S .,
255,
Carroll, S .F .
and Collier, R .J .
(1980) J . Biol .
Chem .
12016-12019 . BOTULINUM NEUROTOSIN AS A PROBE FOR STUDYING NEUROTRANSMITTER RELEASE J .O . DOLLY I , C .K . TSE 1 , J . BLACRI , R . WILLIAMS I , P . HAMBLETON z and J . MELLING z 1 Department of Biochemistry, Imperial College, London and z C .A .M .R .~Porton Down, U .R .
It is known that botulinum toxin, produced by Clostridium botuZi~ttan, irreversibly and specifically inhibits release of acetylcholine from nerve terminals, with unique potency . Unavailability of adequate quantities of the pure neurotozin has forced moat investigators to use its high molecular weight complea(es) with haemagglutinin . Ia thin study, large amounts of neurotozin (Type A) were, therefore, purified to homogeneity and radiolabelled . Thin allowed it to be employed as a probe for nerve membrane components concerned with transmitter release at peripheral and central synapses .
Following isolation of the tozin complea by conventional methods, use of affinity and ion-ezchange chromatography ~ave a good yield (23% of that in the initial culture) of highly active (10 mouse LDsa/mg protein) and homogeneous neurotozin (BoNT) that was completely free of haemagglutinin . It gave a single sharp band on SDS polyacrylamide gel electrophoresis under non-reducing conditions, with a molecular weight of 140,000 ; after reduction equal amounts of two polypeptides (Mr of 99,000 and 55,000) were present . Likewise, one protein band was seen on isoelectric focussing with pI~6 .3 and in double immunodiffuaion gels using horse antibodies against the complex . izs l-Iodination of BoNT to high specific activity (>2,000Ci/mmol) was achieved with good retention of neuro-toxicity ; both subunits were labelled . In vitro application of BoNT to a frog neuromuscular preparation produced near-complete inhibition of transmission, without any detectable poatsynaptic effects . Injection of the BoNT into rat leg muscle produced paralysis ; the frequency
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 .