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Binding properties of beta-bungarotoxin at the neuromuscular junction
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BINDING PROPERTIES OF BETA-HUN(iAROTOXIN AT THE NEUROMUSCULAR JUNCTIOif
C. Caratach, B. Marauda, R. Miledi and P .N . Strong Department of Biophysics, University College London
Beta-bungarotoxin, the most abundant ( by xeight ) of the presynaptically acting neurotoxina in the venom of the krait, BunRarus multicinctus , inhibits the release of acetylcholine at the neuromuscular junction. The toxin is composed of two chains, linked by an essential disulphide bridge . The larger chain ( A chain, MW 13,500 daltons ) shares remarkablq sequence horology with snake venom and pancreatic phoapholipase A enzyres . Ae right be predicted from its amino acid sequence, beta toxin has the enzymati~ properties of a conventional phoapholipase A2 when measured with artificial substrates . Accumulating evidence suggests that the phoapholipase activity is a necessary ( but not sufficient ) requirement for the toxin's ability to irreveraib>ar inhibit neurotranemítter release. Selective modification of one histidine residue with p-bromophenacyl bromide simultaneously the calcium dependent eliminates both phospholipaae activity and neurotoxicity . phosphplipase activity is competitively inhibited by strontium . Heta-toxin inhibits synaptic transmission only when calcium is present in the extracellular bathing fluid ; when calcium ie replaced b~ strontium, ey-naptic transmission ie normal and is no longer inhibited by the toxin. Electron microscopy studies demonstrate that the toxin selectively disrupts the nerve terminal, leaving m~iacle and surrounding Schxann cell membranes unscathed . Other phospholipases A ( pancreatic ; ~a na a enzyme ; Vipers russellü enzyree ) are not To neurotoxic and do not bloc acetylcholine release at equivalent enzyme concentrations . the toxin's enzymatic se].activity for nerve terminal membraneft, we have proposed account (i) Beta-bungerotoxin initially bin9s ( through the B chain? ) to two alternative models . Subsequently, the A chain a protein receptor in the plasma membrane of the nerve terminal . promotes non-selective hydrolysis of the phoepholipida at the phase boundary between receptor boundary lipids and the fluid phoepholipida of the membrane bilayer in the vicinity (ü) No protein receptor is postulated : the highly basic, positively of the binding Bite . charged toxin binds electroatatically to negatively charged phospholipids of the nerve terminal membrane and rigidifies their hydrocarbon tails . Subaec;uent perturbation of membrane structure and enzymatic hydrolysis of nerve terminal phoapholipide in a similar Hoth models assume that nonmanner to (i) leads to inhibition of transmitter release. neurotoxi~ phospholipaees cannot initially bind to the terminal plasma membrane .
fór
We have sought evidence for beta-biangarotoxin binding to cholinergic nerve terminals . In the presence of active enzyme, a wide range of physiological responses mask the primary binding event; in order to simplify our observations, we have examined toxin binding under conditions where we have inhibited enzymatic activity . In electrophyaiological approaches, we have examined the responses of native toxin at the neuromuscular junction in different calcium-free media ( strontium, magnesium ) and have examined the effects of native toxin in calcium solutions at low temperature . To improve our knowledge of the time-resolution of beta-toxin's action we have also studied direct ionophoretic application of the toxin to individual end-plates . Using antibodies prepared against p-bromophenacylbromide-betabungarotoxin, which have been suitably labelled, we have studied the blndinEç of toxin to motor nerve terminals, using both morphological and biochemical technigv~s . These experiments demonstrate that there is an initial phospholipase-independent inhibition of acetylcholine release at the neuromuscular junction, associated xith toxin binding. This implies the presence of presynaptic sites on the nerve terminal membrane which are in some as yet unspecified manner, able to modulate release of acetylcholine from the terminal .
BINDING PROPERTIES OF BETA-HUN(iAROTOXIN AT THE NEUROMUSCULAR JUNCTIOif
C. Caratach, B. Marauda, R. Miledi and P .N . Strong Department of Biophysics, University College London
Beta-bungarotoxin, the most abundant ( by xeight ) of the presynaptically acting neurotoxina in the venom of the krait, BunRarus multicinctus , inhibits the release of acetylcholine at the neuromuscular junction. The toxin is composed of two chains, linked by an essential disulphide bridge . The larger chain ( A chain, MW 13,500 daltons ) shares remarkablq sequence horology with snake venom and pancreatic phoapholipase A enzyres . Ae right be predicted from its amino acid sequence, beta toxin has the enzymati~ properties of a conventional phoapholipase A2 when measured with artificial substrates . Accumulating evidence suggests that the phoapholipase activity is a necessary ( but not sufficient ) requirement for the toxin's ability to irreveraib>ar inhibit neurotranemítter release. Selective modification of one histidine residue with p-bromophenacyl bromide simultaneously the calcium dependent eliminates both phospholipaae activity and neurotoxicity . phosphplipase activity is competitively inhibited by strontium . Heta-toxin inhibits synaptic transmission only when calcium is present in the extracellular bathing fluid ; when calcium ie replaced b~ strontium, ey-naptic transmission ie normal and is no longer inhibited by the toxin. Electron microscopy studies demonstrate that the toxin selectively disrupts the nerve terminal, leaving m~iacle and surrounding Schxann cell membranes unscathed . Other phospholipases A ( pancreatic ; ~a na a enzyme ; Vipers russellü enzyree ) are not To neurotoxic and do not bloc acetylcholine release at equivalent enzyme concentrations . the toxin's enzymatic se].activity for nerve terminal membraneft, we have proposed account (i) Beta-bungerotoxin initially bin9s ( through the B chain? ) to two alternative models . Subsequently, the A chain a protein receptor in the plasma membrane of the nerve terminal . promotes non-selective hydrolysis of the phoepholipida at the phase boundary between receptor boundary lipids and the fluid phoepholipida of the membrane bilayer in the vicinity (ü) No protein receptor is postulated : the highly basic, positively of the binding Bite . charged toxin binds electroatatically to negatively charged phospholipids of the nerve terminal membrane and rigidifies their hydrocarbon tails . Subaec;uent perturbation of membrane structure and enzymatic hydrolysis of nerve terminal phoapholipide in a similar Hoth models assume that nonmanner to (i) leads to inhibition of transmitter release. neurotoxi~ phospholipaees cannot initially bind to the terminal plasma membrane .
fór
We have sought evidence for beta-biangarotoxin binding to cholinergic nerve terminals . In the presence of active enzyme, a wide range of physiological responses mask the primary binding event; in order to simplify our observations, we have examined toxin binding under conditions where we have inhibited enzymatic activity . In electrophyaiological approaches, we have examined the responses of native toxin at the neuromuscular junction in different calcium-free media ( strontium, magnesium ) and have examined the effects of native toxin in calcium solutions at low temperature . To improve our knowledge of the time-resolution of beta-toxin's action we have also studied direct ionophoretic application of the toxin to individual end-plates . Using antibodies prepared against p-bromophenacylbromide-betabungarotoxin, which have been suitably labelled, we have studied the blndinEç of toxin to motor nerve terminals, using both morphological and biochemical technigv~s . These experiments demonstrate that there is an initial phospholipase-independent inhibition of acetylcholine release at the neuromuscular junction, associated xith toxin binding. This implies the presence of presynaptic sites on the nerve terminal membrane which are in some as yet unspecified manner, able to modulate release of acetylcholine from the terminal .