Bungarotoxin, a Pre-Synaptic Toxin with Enzymatic Activity (Neurotoxin/Phospholipase A2/Synaptic Transmission) PETER N
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nat. Acad. Sci. USA Vol. 78, No. 1, pp. 178-182, January 1976 Cell Biology #-Bungarotoxin, a pre-synaptic toxin with enzymatic activity (neurotoxin/phospholipase A2/synaptic transmission) PETER N. STRONG*, JON GOERKEtt, STEPHEN G. OBERG*, AND REGIS B. KELLY*t Departments of * Biochemistry and Biophysics, t Physiology, and the t Cardiovascular Research Institute, University of California, San Francisco, San Francisco, Caoif. 94143 Communicated by Donald Kennedy, October 30, 1975 ABSTRACT ,5-Bungarotoxin, a pre-synaptic neurotoxin naja afid Vipera russelli. Additionally, we observe that ,B- isolated from the venom of the snake Bungarus multicinctus, bungarotoxin can modify synaptic transmission only when has been shown to modify release of neurotransmitter at the have neuromuscular junction. In this communication, we demon- the ionic requirements of the phospholipase activity strate that jP-bungarotoxin is a potent phospholipase A2 (phos- been met. Since we find that other phospholipases at equiva- phatide 2-acyl hydrolase, EC 3.1.1.4), comparable in activity lent concentrations are not toxic, we propose that the phos- with purified phospholipase enzymes from Naja naja and Vi- pholipase activity is a necessary but perhaps not sufficient pera russeii. The phospholipase activity of fl-bungarotoxin facet of fl-bungarotoxin's mode of action. We suggest that requires calcium and is stimulated by deoxycholate. When the phospholipase acts preferentially on pre-synaptic neuro- strontium replaces calcium no phospholipase activity is de- tected. Since neuromuscufar transmission is not blocked nal membranes to modify release of transmitter by altering when calcium is re laced by strontium, it was possible to ex- the probability of fusion of transmitter-containing vesicles amine the effects of the toxin on neuromuscular transmission with the nerve terminal membrane. in the presence of strontium. Under these conditions, when the phospholipase activity should be inhibited, the toxin has little or no effect on neuromuscular transmission. If fl-bun- MATERIALS AND METHODS garotoxin owes its toxicity in part to its enzymatic activity, then it must be placed in a different class from those toxins Reagents. venom from the snake Bungarus multi- which produce their effect by binding passively to an appro- Crude priate receptor. cinctus was obtained from the Ross Allen Reptile Institute, Silver Springs, Fla. Purified phospholipase A enzymes from The release of packaged secretions in response to a sudden Naja naja and Vipera russellhi were the gifts of Dr. J. Sa- demand occurs in many diverse cells (e.g., hormones in en- lach (Veterans Administration Hospital, San Francisco). Soy- docrine glands, digestive enzymes in exocrine glands, and bean phosphatidylcholine (SoyPC) was a gift of Dr. H. Eik- neurotransmitters at nerve endings). It has been suggested ermann (Nattermann and Co., Cologne). Saturated egg that such processes occur by exocytosis-fusion of the mem- phosphatidylcholine (SEPC) was prepared by the catalytic brane of the secretory granule with the cell membrane (1). hydrogenation of purified egg phosphatidylcholine. l-Acyl- The similar calcium requirements of neural and nonneural 2-[3H]acyl-sn-glycero-3-phosphorylcholine ([3H]SEPC), ob- secretory systems (2) have strengthened the idea that many tained by catalytic tritiation of egg phosphatidylcholine, was secretory processes are the same at the molecular level. a gift of Dr. R. Mason (University of California, San Francis- However, the molecular basis for any release system is un- co). known. Purification of ft-Bungarotoxin (Molecular Weight The most well-understood secretory process is probably 21,800). The toxin was purified as previously described (9, the release of acetylcholine at the neuromuscular junction, 13). The purity of i0-bungarotoxin was analyzed by sodium thanks to quantitative electrophysiological measurements dodecyl sulfate polyacrylamide gel electrophoresis (9), iso- (3). Even so, electrophysiology by itself cannot test the mo- electric focusing techniques using broad range (pH 3-10) lecular models that it proposes. In recent years, the discov- Ampholynes (9, 14), and phosphocellulose (Whatman P-li) ery of highly specific neurotoxins which interfere with the ion-exchange chromatography [12 X 0.7 cm diameter 0.3- release of transmitter from nerve terminals [e.g., botulinum 0.7 M potassium phosphate gradient, pH 6.2, modified from toxin (4, 5), black widow spider venom (6, 7), 0-bungarotox- the procedure of Cooper and Reich (15)]. Purified fl-bungar- in (8-10), and notexin (11, 12)] has provided us with poten- otoxin was stored frozen in distilled water or Tris-HCl buff- tially valuable tools for elucidation of the molecular basis of er, pH 7.6 at -20°. The toxin retains full neurotoxicity and neurotransmitter release via biochemical technology. phospholipase activity for many months after repeated In this paper we examine some of the biochemical and freeze-thaw cycles. electrophysiological properties of one such neurotoxin, ,B- Phospholipase A Assay. Purified ,B-bungarotoxin was de- bungarotoxin, which has been shown by electrophysiological salted by overnight dialysis (40, distilled water) prior to measurements to act on the pre-synaptic terminal and to assaying for phospholipase activity. Phospholipase activities have no post-synaptic effects (8, 9). We find that f3-bungaro- were determined using a pH-stat essentially according to toxin is a powerful calcium-dependent phospholipase A2 published procedures (16, 17). As is found for pancreatic (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), comparable in phospholipase A2, activity was greatly enhanced by the pres- activity to purified phospholipase A enzymes from Naja ence of the emulsifying agent sodium deoxycholate (17). For optimal activities, equimolar concentrations of deoxycho- Abbreviations: SoyPC, soybean phosphatidylcholine; SEPC, saturat- late and phosphatidylcholine were required. Substrate [0.2 ed egg phosphatidylcholine; [3H]SEPC, 1-acyl-2-[3H]acyl-sn-glyc- ml of SoyPC in ethanol, 8 ,umol, containing less than 2% lyso ero-3-phosphorylcholine; TLC, thin-layer chromatography; products as determined by thin-layer chromatography m.e.p.p., miniature endplate potential. (TLC)] was added under a constant stream of nitrogen to 4 178 Downloaded by guest on October 2, 2021 Cell Biology: Strong et al. Proc. Nat. Acad. Sci. USA 73(1976) 179 O 0 I z >04 0 0 (A >r m LU -) o C14 m_ Q m < .15 z ::I .10O .05 FRACTION NUMBER 0 5 10 15 20 25 30 FIG. 1. CM-cellulose ion-exchange chromatography of fl-bun- garotoxin. Toxic peak fractions from a CM-Sephadex ion-exchange [,/3 -BUNGAROTOXIN] ,nM column (9) were concentrated and desalted using an Amicon UM-2 filter and then applied to a CM-cellulose column (Whatman CM- FIG. 2. Determination of phospholipase activity of fl-bungaro- 32, 25 cm X 1.6 cm diameter), previously equilibrated with 0.05 M toxin using a pH-stat technique. Dialyzed toxin was added to an NH4OAc (pH 5.0). The column was eluted with a 200 ml linear salt incubation mixture consisting of 8 gtmol of phosphatidylcholine gradient, 0.3 M NH4OAc (pH 5.5) to 0.7 M NH4OAc (pH 6.5) and and 7.5 gmol of sodium deoxycholate in 4 ml of a salt medium (10 2.0 ml fractions were collected. Fractions absorbing at 280 nm were mM CaCl2, 100 mM NaCl, 0.05 mM EDTA). Fatty acid liberated dialyzed against distilled water and the phospholipase activity was in the presence of toxin was titrated to pH 8 (370) with 4 mM measured as described (Fig. 2). All chromatographic procedures NaOH, using an autotitrator. Linear kinetics were observed during and dialysis operations were carried out at 40. the first 5 min after toxin addition and from the initial slope, phos- pholipase activity was computed, expressed as ,ueq of fatty acid liberated per min. ml of a salt reaction mixture (100 mM NaCl, 10 mM CaC12, 0.05 mM EDTA) at 370 in the assay chamber (18). Typically lose column and eluted with a linear NH4OAc salt gradient, 15 ,umol of sodium deoxycholate was introduced and the re- two protein peaks were resolved (Fig. 1). The minor protein action was started by addition of toxin (<3 Mg) in unbuf- component (11%), eluted after the bulk of the material, was fered distilled water. Fatty acid liberated in the incubation less than 2.5% as toxic as the major component, whose mini- cell was titrated with 4 mM NaOH to pH 8.0 (Radiometer mum lethal dose was 0.01 ,g/g of mouse. The peak of phos- model TTT-11 Autotitrator). The rates of SoyPC hydrolysis pholipase activity coincided with the major component; were linear for at least the initial 5 min and it was within from an estimate of the detection limits of our assay, the spe- this time period that reaction rates were computed. Activity cific activity of the minor component must be less than is expressed as the liberation of fatty acid in ,eq/min. Spe- 0.05% of the major component. cific activity is given by the number of ueq of fatty acid lib- In order to be sure that the phospholipase activity of ,3- erated per min/mg of protein. Duplicate assays were repro- bungarotoxin was not due to a contaminant which coelutes ducible within 5%. with the toxin, we had to demonstrate the purity of our sam- Thin-Layer Chromatography. Aliquots from the incubat- ple of ,B-bungarotoxin. The major component of The CM-cel- ed assay mixture were extracted according to the method of lulose column was homogeneous as evidenced by sodium do- Bligh and Dyer (19) and subjected to TLC using solvent sys- decyl sulfate-polyacrylamide gel electrophoresis in the ab- tems previously described (20). Reaction products were sence of reducing agents. In the presence of 2% 2-mercapto- identified after exposure to iodine vapor or under UV light ethanol, two bands were observed (9); these correspond to after spraying with an aqueous solution of 8-anilino-1-naph- the two subunits held together in the native toxin by disul- thalene sulfonic acid (2.5 mg/ml).