Evert Karlsson

Isolation of the nicotinic acetylcholine receptor by biospecific chromatography on insolubilized Naja naja neurotoxin

The electric organs of fishes belonging to the families of Torpedinidae and Gymnotidae are derived from striated muscle and cholinergically innervated. Their high content of “nicotinic” acetylcholine receptors, for Torpedo the number of about 1014 receptors sites per g fresh tissue has been given [ 11, makes them the best source known for purification of cholinergic receptor, but the actual amount of receptors is still very small as compared to that of other material present. An isolation procedure requiring many different purification steps is likely to give a low yield of the purified substance. Biospecific (affinity) chromatography to selectively concentrate and isolate the receptor should therefore be a convenient method. Elapid and hydrophid venoms contain neurotoxins which block the nicotinic acetylcholine receptor, thus mimicking the action of curare [2]. These venom neurotoxins bind to the receptor and the binding is often regarded to be irreversible. However, mice given sublethal doses of neurotoxins (0.8-0.9 X LDloO) and showing severe dyspnea may recover within a few hours [3]. This observation does not seem to be in accordance with an irreversible block. Furthermore, partial dissociation of toxin-receptor complexes and protection against toxin binding by cholinergic substances has been observed in vitro [4-61. We describe here an isolation method for the nicotinic cholinergic receptor based on biospecific chromatography,

The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (australian tiger snake)

Notexin from the venom of Notechis scutatus scutatus is a basic protein with 119 amino acid residues and seven disulfide bridges which blocks neuromuscular transmission by interfering with the release of acetylcholine from the motor nerve terminals [ 1,2] . Notexin exhibits phospholipase activity and is highly homologous to phospholipase A from porcine pancreas and various snake venoms [3-51, raising the possibility that the neurotoxicity is catalytic in nature, involving the hydrolysis of a special phospholipid structure in the nerve terminal. Furthermore, it has recently been suggested that another presynaptic snake venom neurotoxin, /I-bungarotoxin, acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals through a phospholipase A activity [6], The discovery that porcine pancreatic phospholipase AZ can be completely inactivated by the modification of a single histidine residue with p-bromophenacyl bromide [7] appeared to offer a means of assessing the role of phospholipase activity in the presynaptic blocking action of notexin. Notexin exhibited a 99.8% loss of both phospholipase A activity and lethal neurotoxicity upon treatment withp-bromophenacyl bromide. Ultraviolet difference spectroscopy and amino acid analysis showed that the inactivation was accompanied by the incorporation of 1 pbromophenacyl residue and the disappearance of 1 histidine residue per mole of protein. The modified residue was identified as histidine-48,

Purification of a neurotoxin from the venom of Naja nigricollis

A neurotoxin, called toxin α, has been isolated from the venom of Naja nigricollis by ion-exchange chromatography on Amberlite IRC-50. The yield of neurotoxin obtained corresponded to about 3% of crude venom, by weight. n nThe neurotoxin was homogeneous by chromatography on IRC-50, free-zone electrophoresis, starch-gel electrophoresis, immunoelectrophoresis, and end-group analysis. n nThe results of amino acid analysis, end-group analysis, and alkylation studies indicate that the neurotoxin of 61 amino acids in a single chain cross-linked by four disulfide bridges and terminated by leucine and asparagine at its amino- and carboxyl-terminal ends, respectively. The formula weight is 6787. n nThe neurotoxin contains one residue each of tyrosine and tryptophan and its molar extinction coefficient at neutral pH is 8.7·103. n nThe neurotoxin is strongly basic and its cathodal mobility at pH 8.7 and 1° is 5.0·10−5cm2·V−1·sec−1. n nThe L.D.100 of the pure neurotoxin, determined by intravenous injection into mice weighing 18–20 g, is 1.8 μg, as compared with a value of 16 μg for crude venom. n nThe preparation and some properties of the reduced and S-carboxymethylated derivative are described.

Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations

Four polypeptides were isolated by non-exchange chromatography from the crude cardiotoxin fraction of Thailand cobra (Naja naja siamensis) venom. Fraction I and 71 amino acid residues including 1 tryptophan, while fractions, II, III and IV had 60 amino acids and no tryptophan. When tested on isolated skeletal muscle (chick biventer cervicis, chick embryo muscle in culture, guinea pig hemidiaphragm) and cardiac muscle (guinea pig and cat left atria, cat papillary muscle) preparations, fractions II, III and IV, but not fraction I, caused contracture and depolarization. The cardiotoxin-induced contractions could be prevented by pretreatment with raised concentrations of calcium, but were not influenced by a wide range of pharmacological agents which modify nerve-muscle transmission or muscle contractility. The results suggest that cardiotoxins do not act at a specific step in normal excitation-contraction coupling but directly on cell membranes, where they probably cause the formation of pores which result in depolarization and in the influx of calcium.

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin—receptor interaction. The muscarinic antagonist N‐methyl‐[3H]scopolamine was used as a ‘reporter’ ligand. Evidence for a mechanism of toxin—receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin—receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.

Immunoglobulins from patients with amyotrophic lateral sclerosis affect human erythrocyte acetylcholinesterase

Human erythrocyte acetylcholinesterase (AChE) solubilized with Triton X-100 and obtained as a complex with micelles containing Triton and membrane phospholipids was incubated with immunoglobulins (Igs) from patients with amyotrophic lateral sclerosis (ALS) and from normal individuals. The temperature dependence of the AChE activity was determined. Biphasic (broken) Arrhenius plots were obtained with control Igs with the break point at 32.8 +/- 0.3 degrees C (SD, n = 18) indicating that the enzyme changes its conformation at this temperature. With ALS-Igs monophasic (linear) plots were observed in 14 cases and a biphasic in one case. ALS-Igs prevent the conformational change occurring at the break point temperature. The activation energy at physiological temperature increased by 60% from 2.4 to 3.8 kcal/mol (10.0-15.9 kJ/mol) which implies that ALS-Igs inhibit AChE. Thus, ALS-patients have autoantibodies that change the normal behaviour of erythrocyte AChE and which bind to the enzyme molecule or/and to phospholipids associated with the enzyme. At least part of the autoantibodies should be directed against the enzyme molecule, since a change in the Arrhenius plot was also observed in a control experiment with AChE which probably had micelles without any phospholipids. This enzyme was isolated by affinity chromatography and was washed with a buffer containing Triton X-100 before desorption from the affinity column, a treatment known to remove all phospholipids from erythrocyte AChE.

Kinetic evidence for different mechanisms of interaction of black mamba toxins MTα and MTβ with muscarinic receptors

Abstract By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [ 3 H]- N -methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MTα and MTβ, interact with the receptors via kinetically distinct mechanisms. MTβ bound to receptors in a one-step, readily reversible process with the dissociation constant K d =5.3 μM. The binding mechanism of MTα was more complex, involving at least two consecutive steps. A fast receptor–toxin complex formation ( K T =3.8 μM) was followed by a slow process of isomerisation of this complex ( k i =1.8×10 −2 s −1 , half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps ( K T =1.4 μM, k i =8.3×10 −4 s −1 , half-time 840 s). The slow isomerisation process delays the effect of MTα and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.

Polypeptide neurotoxins from mamba venoms that facilitate transmitter release

Abstract As Howard described in his recent TIPS review, most of the prejunctional neurotoxins which are found in snake venoms are phospholipases which destroy motor nerve terminals and hence block transmission to skeletal muscles. However, there is another group of neurotoxins that have recently been isolated from mamba venoms. These toxins, including the prototype dendrotoxin, enhance the amount of chemical transmitter released in response to stimulation. They do not have enzyme activity and do not damage the nerve terminals. Because of their high potency and virtual irreversibility, dendrotoxin and its homologues will be useful in studies of the mechanisms controlling neurotransmitter release and for isolating components involved in release processes.

Immunoglobulin-mediated activity against red blood cells in the saliva of amyotrophic lateral sclerosis (ALS) patients

Immunoglobulin (Ig)‐mediated activity in plasma directed towards normal blood type matched red blood cells (RBC) inducing haemolysis in vitro has earlier been demonstrated to be a characteristic feature in ALS‐patients. In this study, saliva of ALS‐patients, normal and diseased controls was tested with the same in vitro test. An increased degree of haemolysis was induced by the ALS‐patient as compared with control samples. The activity thus found in saliva had the same basic characteristics as that earlier described for plasma; it reacted similarly to serial dilution and was retained in salivary Ig. The effect on red blood cells of saliva from patients with bulbar paralysis was larger than that of saliva from ALS‐patients lacking bulbar symptoms. It is discussed whether cytotoxic Ig in saliva could be pathophysiologically active in bulbar paralysis by means of passage through the oral mucosa and local action on motor end plates in perioral muscles.